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Jun 2013
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In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining
小鼠体内siRNA转染CD40沉默和肾免疫染色评价   

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Abstract

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool.


Graphic abstract:



CD40 siRNA mechanism


Keywords: siRNA (siRNA), CD40 (CD40), CD40L (CD40L), Cholesterol-derived oligonucleotide (胆固醇衍生的寡核苷酸), Kidney (肾脏), siRNA therapy (siRNA治疗), Off-target effects (非目标效应)

Background

The co-stimulatory molecule CD40 and its ligand CD40L are one of the best characterized immune checkpoints involved in the pathophysiology of autoimmune and inflammatory diseases, including cancer, Graft-versus-Host-Disease, inflammatory bowel diseases, systemic lupus erythematosus (SLE), rheumatoid arthritis, type 1 diabetes mellitus, allograft rejection, and atherosclerosis (Lutgens et al; 2010; Ripoll et al., 2013; deRamón et al., 2015; Hueso et al., 2016; Hueso et al., 2019; Karnell et al., 2019). CD40 is a 43-50 kDa transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily and is expressed on the surface of many immune cells. The interaction of CD40 with its ligand CD40L (CD154) induces the trimerization of CD40 and stimulates downstream signaling, including the NF-κB pathway that upregulates proinflammatory genes (Elgueta et al., 2009). Thus, inhibition of the CD40-CD40L dyad is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality, such as the administration of small molecule inhibitors of the interaction of CD40 with TRAF6 (Bosmans et al., 2020), liposome-loaded anti-CD40 antisense oligonucleotides (ASO) (Arranz et al., 2013), anti-CD40 siRNAs (Pluvinet et al., 2004; deRamón et al., 2015; Hueso et al., 2016), or monoclonal antibodies (Remer et al., 2017). Currently, there are three clinical trials testing the safety and efficacy of the anti-CD40 monoclonal antibody, CFZ533, to prevent acute rejection in renal (NCT03663335) or liver (NCT03781414) transplant patients and to evaluate its effects on the kidney in patients with lupus nephritis (NCT03610516). Other clinical trials under development use an anti-CD40L antibody (NCT03605927) to prevent acute Graft-Versus-Host-Disease (NCT03605927), or an oncolytic adenoviral vector that expresses an anti-CD40 antibody (NCT03852511) to treat advanced or metastatic tumors. Our group has developed a chemically stabilized, cholesterol-conjugated small inhibitory RNA molecule against murine CD40 and reported the evaluation of its potency, distribution, and durability of effects following systemic administration (Ripoll et al., 2013; deRamón et al., 2015, Hueso et al., 2016, Hueso et al., 2019). Here, we summarize a protocol describing the systemic administration of this specific anti-CD40 siRNA in different mouse models.


Materials and Reagents

  1. Consumables

    1. Disposable gloves

    2. RNAase-free 1.5 ml polypropylene tubes

    3. 96-well plates

    4. 0.5 ml polypropylene tubes

    5. 50 ml Falcon conical tubes (Corning, catalog number: 45352054)

    6. 70 μm nylon cell strainers (BD Biosciences , catalog number: 45352350)

    7. Cell culture plates

    8. Syringes

    9. Towels

    10. Sample collection tubes

    11. 23-25 G needles

    12. 2 ml RNAase-free Eppendorf tubes (Merck KGaA, catalog number: T2795)

    13. Poly-L-lysine-coated slides (Merck KGaA, catalog number: P0425)

    14. Whatman No. 1 filter paper


  2. Reagents and Kits

    1. Nuclease-free water (DEPC-treated water)

    2. Absolute isopropanol (Merck KGaA, catalog number: I9516)

    3. Tris base (Tris-hydroxymethyl-aminomethane; Bio-Rad Lab, catalog number: 161-0719)

    4. 3 M sodium acetate (Merck KGaA, catalog number: S7899)

    5. Bromophenol Blue-Xylene Cyanole Dye Solution (Merck KGaA, catalog number: B3269-5mL)

    6. N,N,N’,N’-tetramethylethylenediamine, TEMED (Bio-Rad, Hercules, catalog number: 161-0800)

    7. Amonium persulfate, APS (Bio-Rad, Hercules, catalog number: 161-0700)

    8. Methanol (Merck KGaA, catalog number: 82762)

    9. Absolute ethanol (Obtained from general chemical providers)

    10. Hydrogen peroxide (Obtained from general chemical providers)

    11. D+ sucrose (AppliChem GmbH, catalog number: 200-334-9)

    12. Ethylenediaminetetraacetic (EDTA, Merck KGaA, catalog number: E6758)

    13. RNase-ZAP (Merck KGaA, catalog number: R2020)

    14. BHT (Butylated hydroxytoluene; Merck KGaA, catalog number: W218405)

    15. FACS lysing solution (Becton Dickinson, catalog number: 349202)

    16. Xylene (mixture of isomers, VWR International, catalog number: 28975.360)

    17. DPX mounting medium (VWR International, catalog number: 360294H)

    18. Jelly from porcine skin (Sigma-Aldrich, catalog number: G1890)

    19. Oil-Red O (Merck KGaA, catalog number: O0625)

    20. RPMI 1640 medium (Biological Industries)

      Note: Reagents 1-20 were stored at room temperature.


    21. Opti-MEM (Themo Fisher, catalog number: 51985034)

    22. Fetal bovine serum (Lonza Pharma&Biotech, catalog number: 14-802F)

    23. Albumin ELISA kit (Active Motif)

    24. DEPC (Diethyl Pyrocarbonate; Merck KGaA, catalog number: D5758).

    25. 40% acrylamide/Bis 29:1 (Bio-Rad, Hercules, catalog number: 161-0146)

    26. Propidium iodine (StemCell Technologies, catalog number: 75002)

    27. TRIzol (ThermoFisher Scientific, catalog number: 15596026)

    28. DAB substrate (3,3’Diaminobenzidine tetrahydrochloride hydrate, Sigma-Aldrich, catalog number: D5637-5G)

    29. Flow cytometry staining buffer (ThermoFisher Scientific, catalog number: 00-4222)

    30. 100 mM Penicilin/Streptomycin (ThermoFisher Scientific, catalog number: 15070-063)

      Note: Reagents 21-30 were stored at 4°C.


    31. 200 mM L-glutamine (ThermoFisher Scientific, catalog number: 25030-024)

    32. High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, catalog number: 4368814)

    33. PureLinkTMRNA Mini kit (ThermoFisher Scientific, catalog number: 12183020)

    34. 5’ RACE system for rapid amplification of cDNA ends kit (ThermoFisher Scientific)

    35. Oligofectamine 2000 (ThermoFisher Scientific, catalog number: 12252011)

    36. GM-CSF (R&D systems, catalog number: 215-GM)

    37. Lipopolysaccharides (LPS) from E. coli Serotype O111:B4 (Sigma-Aldrich, catalog number: L4391)

    38. GeneEraser Luciferase Suppression-Test System (Stratagene/Agilent, catalog number: 240192)

    39. Luciferase Assay System (Promega, catalog number: E1531)

    40. Polyfect transfection reagent (Qiagen, catalog number: 301105)

    41. Serum normal goat (Merck KGaA, catalog number: G9023) or horse 20% (Merck KGaA, catalog number: H0146)

      Note: Reagents 31-41 are stored at -20°C.


    42. Taqman gene expression assays (ABI/ThermoFisher Scientific) (Table 1)


      Table 1. Commercial TaqMan probes used in this study

      Name
      of the gene tested


      Probe code1

      Gene unique identifier2

      Position3

      CD40

      Mm00441891_m1

      NM_011611

      121

      CD40L

      Mm00441911_m1

      NM_011616

      348

      IL1b

      Mm01336189_m1

      NM_008361

      55

      NLRP3

      Mm00840904_m1

      NM_145827

      499

      Apelin (Apln)

      Mm00443562_m1

      NM_013912

      414

      C3

      Mm01232779_m1

      BC029976

      517

      CD55

      Mm00438377_m1

      NM_010016

      1154

      FOXP3

      Mm00475165_m1

      NM_001199347

      1307

      IL6

      Mm99999064_m1

      NM_031168

      233

      IL10

      Mm00439614_m1

      NM_010548

      232

      MCP1 (Ccl2)

      Mm00441242_m1

      NM_011333

      165

      RANTES (Ccl5)

      Mm01302428_m1

      NM_013653

      245

      TLR3

      Mm00628112_m1

      NM_126166

      280

      TLR4

      Mm00445273_m1

      NM_021297

      112

      TLR9

      Mm00446193_m1

      NM_031178

      106

      AIM2

      Mm01295719_m1

      NM_001013779

      869

      18S rRNA

      Hs99999901_s1

      X03205

      604

      (1) Commercial code of the Taqman probes used (ThermoFisher Scientific).

      (2) Refseq/Genbank unique identifiers of the genes tested.

      (3) Base position contained within the probe.


    43. 0.1 M citrate buffer, pH 6 (see Recipes), stored at room temperature

    44. 0.01 M citrate buffer (see Recipes), stored at room temperature

    45. Oil-Red working solution (see Recipes), stored at room temperature

    46. Phosphate-buffered saline (PBS), pH 7.5 (see Recipes), stored at room temperature

    47. PBS-Triton (PBST) (see Recipes), stored at room temperature

    48. PS buffer (see Recipes), stored at room temperature

    49. Complete culture medium (see Recipes), stored at 4°C

    50. Secondary antibodies (for a 1:200 dilution) (see Recipes), keep at -20°C

    51. Annealing buffer 10× (see Recipes)

    52. DEPC-treated water (see Recipes)

    53. 1 M Tris-HCl, pH 7.5 (see Recipes)

    54. 12% acrylamide gel (see Recipes)

    55. Non-denaturing gel loading buffer (see Recipes)

    56. 4% PFA solution in PBS (see Recipes)

    57. 0.3% Oil-Red O stock solution (see Recipes)

    58. Serum normal goat or horse 20% (see Recipes)


  3. Primary antibodies

    In this work we used the following specific primary antibodies:

    1. Rabbit polyclonal anti-CD40 antibody (C-20, Santa Cruz Biotech, catalog number: sc-975)

    2. Rabbit polyclonal anti-CD154 antibody (H215, Santa Cruz Biotech, catalog number: sc-9097)

    3. Mouse monoclonal anti-DC-SIGN antibody (1B10, Santa Cruz Biotech, catalog number: sc-23926)

    4. Rabbit polyclonal anti-NF-κB p65 antibody (anti-phospho-S536, Abcam, catalog number: Ab86299)

    5. Anti-F4/80 antibody (Hycult Biotech, catalog number: HM1066)

    6. Rabbit anti-mouse collagen-IV antibody (Chemicon international, catalog number: AB756p)

    7. Goat polyclonal anti-human C3c antibody conjugated to FITC (Nordic-MuBio, catalog number: GAHu/C3c/FITC)


      Furthermore, the following antibodies from BD Biosciences (San Jose, CA, USA) were used for flow cytometry:

    8. Anti-CD11c antibody (clone HL3)

    9. Anti-CD11b antibody (clone M1/70)

    10. Anti-CD40 antibody (clone HM40-3)

    11. Anti-CD80 antibody (clone 16-10A1)

    12. Anti-CD86 antibody (clone GL1)

      Note: All primary antibodies were stored at -20°C, unless another temperature was specifically stated by the manufacturer.


  4. Secondary antibodies and histological reagents

    The following secondary antibodies were used in this work:

    1. Alexa 488-labeled chicken anti-goat (ThermoFisher Scientific, catalog number: AB_2535870)

    2. Goat anti-rabbit (ThermoFisher Scientific, catalog number: AB_2633280)

    3. Alexa 555-labeled goat anti-mouse (ThermoFisher Scientific, catalog number: AB_2535844)

    4. Alexa 546-labeled goat anti-rabbit (ThermoFisher Scientific, catalog number: AB_2633280)

    5. Unconjugated goat anti-rat (Novus Biologicals)

    6. Biotinylated horse anti-goat (Vector Laboratories, catalog number: BA-9500)

    7. FITC-conjugated goat anti-mouse (Merck KGaA, catalog number: F0257)

    8. VECTASTAIN Elite ABC HRP kit (Vector Laboratories, catalog number: PK-6100)

    9. Avidin/Biotin blocking kit (Vector Laboratories, catalog number: PK-4001-NB)

      Note: All secondary antibodies were stored at 4°C, unless another temperature was specifically stated by the manufacturer.


      The following histological reagents were used:

    10. Harris hematoxylin solution (Sigma-Aldrich, catalog number: HHS32-1L)

    11. Eosin Y solution, alcoholic (Sigma-Aldrich, catalog number: HT110132-1L)

    12. Periodic acid (Sigma-Aldrich, catalog number: P0430-25G)

    13. Schiff reagent (Sigma-Aldrich, catalog number: 3952016-500ML)

    14. PAS staining kit (Sigma-Aldrich, catalog number: 101646)

    15. Oil Red-O reagent (Sigma-Aldrich, catalog number: O0625-100G)

    16. Tissue Tec OCT inclusion compound (Sakura FinetekEurope)

    17. UltraCruz Tm mounting medium (Santa Cruz Biotech, catalog number: sc-24941)

    18. DRAQ5 (ThermoFisher Scientific, catalog number: 65-0880-92)

    Notes:

    1. Used for nuclear counterstaining.

    2. All histological reagents were stored at room temperature.


  5. Animals

    1. Six-month-old NZB/NZW (F1) female mice (The Jackson Lab, Charles River, Wilmington, MA, USA)

    2. Six to eight-week-old male ICR mice (The Jackson Lab, Charles River, Wilmington, MA, USA)

    3. Eight-week-old ApoE-/- female mice (The Jackson Lab, Charles River, Wilmington, MA, USA)

      Note: Animals were housed in a room maintained at a constant temperature and given free access to water and a standard laboratory diet. To accelerate atherosclerosis, ApoE-/- mice were fed a Western diet that contained 0.2% cholesterol and provided 42% of the energy as fat (TD.88137; Harlan-Tekland, Madison, WI, USA). Animals were euthanized by inhalation of isoflurane. Protocols were approved by the Ethics Committee for Animal Research of UB-Bellvitge, and experiments were performed in accordance with the European legislation on Laboratory Animal Experiments.


  6. siRNA oligonucleotides

    In this work, we used a CD40-specific ds-siRNA homologous to both the mouse and rat CD40 mRNA sequence (herein antiCD40-siRNAChol) and a scrambled sequence (s/s) ds-siRNA as the control (herein s/s-control siRNAChol). Both were modified by conjugating a cholesterol (chol) molecule to the 3’ end of the sense strand by means of a pyrrolidine linker.

    The siRNA sequences were as follows:

    Anti-CD40 sense strand: 5’-GUGUGUUACGUGCAGUGACUU-3’

    Anti-CD40 antisense strand: 5’-GUCACUGCACGUAACACACTG-3’

    (s/s), control siRNA sense strand: 5’-ACUACAAGACUCGUGACCAUU-3’

    (s/s), control siRNA antisense strand: 5’-UGGUCACGAGUCUUGUAGUUU-3’

    To determine the transfection efficiencies and organ distributions of the siRNAs, a cholesterol-conjugated and an unmodified anti-CD40 siRNA were labeled with Cy5.5.

    Note: All oligonucleotides were obtained from Microsynth AG (Balgach, Switzerland) and stored at -20°C.


  7. Cell lines

    1. The highly transfectable human embryonic kidney 293FT cell line (ThermoFisher Scientific, catalog number: R70007)

    2. Primary dendritic cells (obtained from the bone marrow of ICR mice)

Equipment

  1. Cell scrapers (Sarstedt AG & CO, Nümbrecht, GE, catalog number: 83.3950)

  2. Scalpels

  3. Forceps: Straight, serrated-tip forceps; straight or curved, serrated-tip fine forceps; and straight fine-tip forceps

  4. Scissors: Straight, blunt scissors; straight, sharp, fine scissors; and micro-dissecting spring scissors

  5. Automatic pipettes (1-10 μl, 20-200 μl)

  6. Hemocytometer

  7. Water bath

  8. Jasco V-650 spectrophotometer (Easton, MD, USA, used to determine the melting point of siRNA duplexes)

  9. Olympum autoanalyzer AU400 (Hamburg, Germany, used to determine urinary protein and creatinine concentrations)

  10. TaqMan real-time PCR ABI Prism® 7700 (ThermoFisher Scientific, used for the qPCR experiments)

  11. BD FACS Canto II cytometer (BD Biosciences, used in the flow cytometry experiments)

  12. Zeiss SteREOLumar V12 microscope (Carl Zeiss AG, used for microscopy experiments)

  13. Leica TCS-SL spectral confocal microscope (Leica Camera AG, used for microscopy experiments)

  14. TD-20/20 luminometer (Turner Designs, used for luciferase assays)

  15. T-25 ULTRA-TURRAXTM homogenizer (IKA®-Werke GmbH & Co)

  16. NanoDrop 2000c spectrophotometer (ThermoFisher Scientific)

  17. Tabletop centrifuge (Eppendorf Cooled Centrifuge, mode: 5424R)

  18. Cell culture CO2 incubator (NuAire Autoflow NU 8700)

Software

  1. Leica confocal software (Leica Camera AG, Wetzlar, Germany)

  2. Image analysis software ProgResCapturePro 2.7.7 (JenoptiK AG, Jena, GE)

  3. ImageJ v1.48 (NIH, Bethesda, MD, USA)

  4. ExpressionSuite software v1.0 or later (ABI, ThermoFisher Scientific, Waltham, MA, USA)

  5. SDS software v2.4 (ABI, ThermoFisher Scientific, Waltham, MA, USA)

  6. FACS DIVA software (BD Biosciences, San Jose, CA, USA)

Procedure

  1. Synthesis and preparation of siRNAs

    Requirements: Disposable gloves, heated water bath, microcentrifuge, automatic pipettes (1-10 μl, 20-200 μl), Jasco V-650 spectrophotometer, RNAase-free 1.5-ml polypropylene tubes, ice, nuclease-free water, annealing buffer (10 mM Tris pH 7.5, 20 mM NaCl), 3 M sodium acetate (pH 5.2), isopropanol, 70% ethanol, oligonucleotides.

    1. Design of anti-CD40 oligonucleotides.

      Analyze the target mRNA sequence (herein murine CD40, NCBI accession X60592.1) to select a number of sequences that comply with the following structure, 5’-GN17C-3’. Nine different sequences were generated to select an optimal target site (Pluvinet et al., 2004). General rules to improve the effectivity of siRNA silencing are: 1) presence of G/C at the 5’ end of the sense strand; 2) presence of A/U at the 5’ end of the antisense strand; 3) presence of at least 5 A/U residues in the first 7 bases of the 5’ end of the antisense strand; 4) no runs of more than 9 G/C residues should be allowed in the sequence (Ui-Tei el al., 2004); 5) secondary structure at the target site should be kept to a minimum (Bohula et al., 2003; Kretschmer-Kazemi Far and Sczakiel., 2003).

    2. Annealing of oligonucleotides to generate ds-siRNAs.

      For oligonucleotide annealing, mix equimolar amounts of complementary sense and antisense strands of the different anti-CD40 and control siRNAs by mixing:

      Oligonucleotide 1/sense strand (100 pmol/µl)………………………………………10 µl

      Oligonucleotide 2/antisense strand (100 pmol/µl)……..…...……………………10 µl

      2x oligo annealing buffer (10 mM Tris, pH 7.5, 20 mM NaCl)…………………50 µl

      Nuclease-free water (up to) ………………………………………..………………………100 µl

      1. Heat for 3 min at 90°C in a water bath and switch it off.

      2. Let the tubes cool down slowly in the water bath to room temperature (<60 min). The final concentration should be 100 pmol/µl ds-siRNA.

      3. Store the annealed ds-siRNA oligonucleotides at 4°C for immediate use or at -20°C for longer storage.

    1. Determination of the melting temperature (Tm) of the ds-siRNAs.

      Heat samples in the Jasco V-650 spectrophotometer using a linear temperature ramp of 0.5°C/min. Melting temperature is obtained as the maximum of the first derivative. The melting point of the unmodified CD40 ds-siRNA is 79°C and that of the cholesterol-derived CD40 ds-siRNA (antiCD40-siRNAChol) is 71°C.

    1. Alcohol precipitation of ds-siRNAs.

      1. Add a 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol, mix.

      2. Keep on ice for 5 min and spin down at the top speed in a microcentrifuge for 10 min.

      3. Carefully aspirate the supernatant, wash the pellet with 0.5 ml cold 70% ethanol, and carefully remove all ethanol.

      4. Air-dry the pellet for no longer than 15 min at room temperature and resuspend the ds-siRNA in nuclease-free water in 2-5-times the original volume.

      5. Quantitate the siRNA concentration using a NanoDrop or equivalent. The final concentration should be 20-50 pmol/µl.

      6. Analyze the siRNA on a non-denaturing 12% polyacrylamide gel (see Recipes) for size and integrity, and store at -20°C or -70°C.

        1. Mix up to 5 μl oligonucleotides with 2 μl loading buffer (see Recipes).

        2. Load the sample on a non-denaturing 12% polyacrylamide gel and subject to electrophoresis at 200-250 V.

        3. Stop electrophoresis when the Bromophenol Blue dye front has migrated two-thirds of the way down the gel.

        4. Stain the gel for 2-5 min in a 1 μg/ml solution of ethidium bromide.

        5. Soak the gel for 2-5 min in water.

        6. Visualize the siRNA using a UV transilluminator.


        The siRNA should migrate as a 21-22 bp band that runs slightly behind the Bromophenol Blue dye front. A second, less intense band running behind the primary siRNA band may be apparent and represents one partially digested strand of siRNA. The underdigested RNA strand is 27 nt in length and does not create any non-specific effects when used to transfect cells.


  2. Determination of the silencing ability of anti-CD40-siRNAChol using the GeneEraser Luciferase Suppression-test system

    Requirements: Disposable gloves, 96-well plates, automatic pipettes (1-10 μl, 20-200 μl), cell scrapers (Sarstedt AG & CO, Nümbrecht, GE, catalog number: 83.3950), microcentrifuge, ice, 0.5-ml polypropylene tubes, pTarget-luc-rCD40, Oligofectamine, HEK-293 cells, Polyfect transfection reagent, 70% ethanol, 1× phosphate-buffered saline (PBS), 1× lysis reagent, luciferase assay reagent, TD-20/20 luminometer.

  1. Generate the apTarget-luc-CD40 plasmid.

    A blunt-end 506 bp fragment corresponding to the partial rat CD40 cDNA sequence (coding sequence of nucleotides 41-547; GenBank Acc. No AF241231) was blunt-end cloned into the plasmid pTarget-luc at the 3'UTR of the luciferase gene.

  2. Transfect HEK-293 cells with siRNAs (100 nM).

    1. Prepare a 100 nM solution of the ds-siRNAs, transfect confluent HEK-293 cells using Oligofectamine according to the manufacturer’s instructions.

    2. After 6 h, transfect the above cells with 400 ng pTarget-luc-CD40 using Polyfect transfection reagent according to the manufacturer’s instructions. Normalize the transfection efficiency by co-transfecting 500 ng pCMV-bGal.

    3. Incubate the cells for 48 h at 37°C in a humidified atmosphere with 5% CO2.

  3. Prepare cell lysates for analysis.

    1. Remove the growth medium from the cultured cells.

    2. Rinse the cells in 1× PBS and remove as much as possible.

    3. In a 96-well plate, dispense 20 µl/well 1× lysis reagent (1× lysis reagent is prepared by adding 4 vol. water to 1 vol. 5× lysis reagent from the kit).

    4. Scrape the cells from the dish and transfer the solution to a microcentrifuge tube.

    5. Pellet the cell debris by brief centrifugation and transfer the supernatant to a new tube.

    6. Mix 20 µl cell lysate with 100 µl luciferase assay reagent and measure the light in the TD-20/20 luminometer.


  1. Bone marrow-derived dendritic cell (DC) extraction, culture, activation, and transfection with siRNAs in vitro

    Requirements: Disposable gloves, 50-ml Falcon conical tubes (Corning, catalog number: 45352054), 70-μm nylon cell strainers (BD Biosciences, catalog number: 45352350), autoclaved materials (forceps, scalpels, scissors), cell culture hood, ice, mice femurs and tibiae, hemocytometer, cell culture plates, sample collection tubes, Oligofectamine 2000, BD FACS lysing solution, complete RPMI 1640 medium (RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 µM L-glutamine, 10% heat-inactivated FBS, and 20 ng/ml GM-CSF), Opti-MEM medium, propidium iodine (stock solution: 1.5 mM), siRNAs, LPS, cytometer.

    1. Euthanize ICR mice according to the institutional guidelines and extract the femurs and tibiae.

      1. Place each mouse onto a sterile surgical pad in a sterile hood.

      2. Spray the mouse with 70% ethanol to avoid contamination of the samples.

      3. Soak the femurs and tibiae in RPMI 1640 medium.

    2. Harvest the bone marrow (BMC).

      Note: All work should be performed under sterile conditions in a cell culture hood.

      1. Cut the femurs at both ends with sterile scissors.

      2. Transfer the bones to RPMI 1640 in a sterile Petri dish.

      3. Vigorously flush the inside of the bones with 1 ml ice-cold RPMI 1640 medium using a 1-ml pipette. Flush 2-3 times until the bones are completely white.

      4. Flush the bone marrow out onto a 70-μm nylon cell strainer placed in a 50-ml Falcon conical tube.

      5. Smash the bone marrow through the cell strainer using a 5-ml plunger, and wash the strainer with 5 ml RPMI.

      6. Centrifuge the cells at 250 × g for 8 min at 4°C and discard the supernatant.

      7. Resuspend the cell pellet in 1 ml RBC lysis buffer and incubate for 5 min at room temperature.

      8. Neutralize the lysis buffer by adding 5 ml FBS.

      9. Centrifuge the cells at 250 × g for 8 min at 4°C, discard the supernatant, and resuspend in 5 ml RPMI 1640 medium. Take out an aliquot of cell suspension to count.

    3. Determine the total number of cells obtained.

      a. Dilute 10 μl bone marrow suspension in 90 μl 1× BD FACS lysing solution (see above).

      b. Count 10 μl cell suspension using a hemocytometer.

    4. Grow 5 × 106 cells.

      Grow cells in 5 ml complete RPMI 1640 medium (see above) supplemented with 10 ng/ml IL-4 and 20 ng/ml GM-CSF in T25 flasks at 37°C in a humidified atmosphere with 5% CO2.

      After 24 h, some of the cells adhere to the plate but many others are suspended in the culture medium. Colonies appear after 72 h. The number of adherent cells gradually decreases over time and on day 5, the suspended cells with dendritic protrusions gradually increase. On day 7, the suspended cells begin to aggregate and the protrusions elongate.

    5. Change the culture medium on days 3 and 5.

      1. Remove the medium carefully so as not to disturb the growing cells.

      2. Briefly centrifuge (250 × g for 8 min) the removed volume of media since DCs are only loosely adherent. Resuspend the cell pellet in the same flask with 10 ml fresh RPMI 1640 supplemented with 20 ng/ml GM-CSF.

    6. Harvest BMDCs (day 8)

      1. Collect non-adherent cells by gently pipetting the culture medium, and transfer into sterile 50-ml centrifuge tubes. Discard the adherent cells that include macrophages.

      2. Loosely adherent BMDCs become easily dislodged into suspension by this process, while macrophages remain adhered to the Petri dish.

      3. Count the total number of cells obtained.

    7. Transfect with siRNAs on day 8.

      Incubate 1 × 106 immature BMDCs/ml with 2 µM unmodified siRNA-Cy5.5 or Chol-siRNA-Cy5.5 (see Section E of the Materials and Reagents section, above) using the cationic lipid Oligofectamine 2000 in Opti-MEM medium on 6-cm dishes.

    8. Treat immature BMDCs with 100 ng/ml LPS on day 9 for 12 h.

    9. Harvest BMDCs

      1. Collect all the cells (detach cells with a scraper).

      2. Centrifuge the cells at 250 × g for 8 min.

      3. Remove the supernatant and resuspend the cell pellet in 5 ml PBS (wash 1)

      4. Centrifuge the cells at 250 × g for 8 min.

      5. Remove the supernatant and resuspend the cell pellet in 5 ml PBS (wash 2)

      6. Centrifuge the cells at 250 × g for 8 min.

      7. Resuspend the cell pellet in 1 ml RPMI 1640 supplemented with 10% FBS and 20 mM penicillin/streptomycin.

      8. Count the cells.

    10. Stain the DCs with a final concentration of ≤1 μg/ml propidium iodide (PI).

      1. Prepare a 1 mg/ml (1.5 M) stock solution by dissolving solid PI in PBS. Protect from prolonged exposure to light.

      2. Whenever possible, prepare and use the stock solution on the same day. If the stock solution must be made in advance, aliquot and store in tightly sealed vials at -20°C (generally stable for up to 1 month).

      3. Resuspend 50 μl cell suspension in 50 μl flow cytometry staining buffer.

      4. Add 5 μl PI staining solution per 100 μl cell suspension. Do not wash the cells after the addition of PI.

      5. Incubate for 5–15 min on ice in the dark.

      6. Analyze the samples immediately by flow cytometry.

    11. Characterize the cell phenotype by flow cytometry.

      1. Block non-specific Fc-mediated interactions with 0.5 μg anti-mouse CD16/CD32 per 100 μl for 15 min at 4°C before staining.

        Antibody-binding kinetics are temperature dependent. Staining on ice may require longer incubation times.

      2. Aliquot 50 μl cell suspension to each tube (one per antibody).

      3. Combine the cell suspension with primary antibodies (the isotype control and the following antibodies: anti-CD11c (clone HL3), anti-CD11b (clone M1/70), anti-CD40 (clone HM40-3), anti-CD80 (clone 16-10A1), and anti-CD86 (clone GL1)) and the appropriate volume of flow cytometry staining buffer to reach a final staining volume of 100 μl. Vortex.

      4. Incubate for at least 30 min on ice. Protect from light.

      5. Wash the cells by adding 2 ml flow cytometry staining buffer to each tube.

      6. Repeat the wash step twice by centrifuging the cells at 250 × g for 8 min at room temperature.

      7. Remove the supernatant and resuspend the pellet in 100 μl flow cytometry staining buffer.

      8. Transfer the cell suspension to polypropylene tubes for flow cytometry.

      9. For storage of the samples before analysis, add 100 μl IC fixation buffer.

      10. Analyze the cells according to fluorescence intensity of the above markers and acquire representative images for subsequent analysis/quantitation.


  2. Evaluation of the silencing activity of antiCD40-siRNAChol in in vivo mouse models

    Requirements: Gloves, scalpels, scissors, syringes, towels, cotton, sample collection tubes, 23–25 G needles, mice, siRNAs, PS buffer (see Recipes), isoflurane, isoflurane chamber, portable liquid nitrogen container, liquid nitrogen, and blood collection tubes.

    1. Separate the mice into treatment and control groups.

      1. Study the in vivo effect of the systemic administration of chol-siRNAs: Use 6-8-week old male ICR mice (4 mice per group). Administer 50 µg antiCD40-siRNAChol or s/s-control siRNAChol via i.p. injection (10 ml/kg in 0.2-µm-filtered PBS). At predefined time points (days 0, 1, 3, 5, 7, and 9), administer single doses of 5 µg LPS from E. coli via i.p. injection. Euthanize animals 4 h post-administration of LPS.

      2. For the lupus nephritis study with 5-month-old NZB/NZW F1, set up the following groups:

        1. CYP: Administer 50 mg/kg i.p. every 10 days (n = 9).

        2. CTLA4: Administer 50 μg abatacept (Orencia, Bristol Myers Squibb) three times weekly (n = 9).

        3. siCD40-1w: Administer 50 μg anti-CD40-siRNAChol i.p. once weekly (n = 9).

        4. siCD40-2w: Administer 50 μg anti-CD40-siRNAChol i.p. twice weekly (n = 9).

        5. Control: Administer 50 μg s/s-control siRNAChol i.p. twice weekly (n = 15).

      3. For the atherosclerosis study with 8-week-old female ApoE-/- mice. Treat the mice twice weekly i.p. with 50 μg anti-CD40-siRNAChol, s/s-control siRNAChol, or vehicle (5 mice per group). Euthanize the animals at 8 weeks (basal group), 10 weeks, 14 weeks, or 24 weeks.

    1. Tissue collection: urine and venous blood.

      1. Place the animals in individual metabolic cages with water and the usual diet, and collect 24 h urine specimens before the onset of treatment and thereafter at monthly intervals.

        Note: If mice do not produce urine in the given time, repeat the procedure on the following day in a warmer room.

      2. Centrifuge the urine samples at 500 × g for 10 min. Collect the urine and retain the sediment for further studies; store the urine and sediment at -20°C.

      3. Determine the weight of the mice twice monthly.

      4. On a monthly basis, perform a non-terminal venous blood collection from the tail vein (without replacement of fluids) using short-term anesthesia by isoflurane inhalation.

        Important points to remember for animal comfort:

        1. Make the animal comfortable by maintaining the temperature at 24-27°C.

        2. DO NOT rub the tails from the base of the tip as this will result in leukocytosis. If the vein is not visible, dip the tail into warm water (40°C).

        3. Insert a 23-25 G needle into the blood vessel and collect blood using a capillary tube or syringe with a needle. In case of difficulties, cut a minimal surface of the skin, prick the vein with a bleeding lancet or needle and collect blood with a capillary tube or a syringe with a needle.

        4. DO NOT try to collect blood more than three times. The maximum collection volume should be 0.2 ml blood per mouse. The estimated blood volume in an adult animal is 55-70 ml/kg body weight (Parasuraman et al., 2010).

        5. After completing blood collection, stop the bleeding using pressure.

        6. Wash the restraint frequently to avoid pheromonal-induced stress or cross infection.

      5. At the end of the study, extract arterial blood by cardiac puncture.

        1. Perform terminal anesthesia of animals in an isoflurane chamber.

        2. Open the chest (thoracotomy) and obtain a blood sample directly from the ventricle, taking care not to collapse the heart.

    1. Tissue collection: liver and kidneys

      1. Euthanize all mice by inhalation of isoflurane.

      2. Dissect out the kidneys and liver through the abdomen and wash them by perfusing ice-cold 1× PBS via the left ventricle.

      3. Resect the kidneys. One kidney will be used for histological evaluation and the other for total RNA extraction.

      4. For histological analysis, remove the upper third of the kidney to ensure that both cortical and juxtamedullary glomeruli are present and fix the piece in 5 ml PS buffer at 4°C for 24-48 h.

      5. For immunofluorescence analysis, place a third of the kidney piece (to ensure that both cortical and medullary glomeruli are present) into the tissue mold and coat in OCT. Place it onto dry ice to freeze and store at -80°C.

      6. For protein and RNA extraction, place 3 × 2 mm3 pieces of kidney cortex and liver into 0.5 ml plastic tubes and snap freeze them in liquid N2. Store at -80°C. For long-term tissue storage for RNA extraction, place the tissue in five volumes of RNA stabilization reagent and store at -80°C.

    1. RNA extraction and purification from kidney tissue.

      Requirements: Disposable gloves, laboratory fume hood, RNase-free pipettes and pipette tips, sterile plasticware, cold TRIZolTM, rotor-stator homogenizer (ULTRA-TURRAXTM), ice, round-bottomed RNase-free tube for tissue homogenization, RNase-free water (water treated with DEPC, see Receips), tabletop centrifuge, 2 ml RNAase-free Eppendorf tubes, NanoDrop, PureLink RNA mini kit, chloroform, 70% ethanol (100% ethanol mixed with RNAse free water), vortex.

      1. Add 1 ml cold TRIzol to 50-100 mg renal tissue in a round-bottomed RNase-free tube, homogenize on ice. Avoid foaming by keeping the tip of the probe submerged in the lysis solution while holding the tip against the tube wall.

      2. Incubate for 5 min at room temperature to allow complete dissociation of the nucleoprotein complex.

      3. Add 0.2 ml chloroform and vortex.

      4. Incubate for 2-3 min at room temperature.

      5. Centrifugate the sample for 15 min at 12,000 × g, 4°C.

      6. The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. Carefully transfer the aqueous phase to a 2-ml Eppendorf tube (important: avoid transferring any of the interphase or organic layer).

      7. Add one volume 70% ethanol and vortex.

      8. Transfer ≤700 μl sample to the spin cartridge and purify total RNA according to the kit instructions (in this case the PureLink RNA mini kit).

      9. Quantify the total RNA using the NanoDrop.

    1. First strand DNA synthesis (reverse transcription).

      Requirements: Ice, RNase-free water, tabletop centrifuge, Eppendorf tubes, High-Capacity cDNA Reverse Transcription kit, thermal cycler, 70% ethanol, vortex.

      The reverse transcription reaction is performed according to the instructions of the kit used (in this case the High-Capacity cDNA Reverse Transcription) using 500 ng RNA.

    2. Determine CD40 mRNA expression in the kidney and liver by qPCR.

      Set up reactions with CD40 primers (Mn_00441891_m1; Taqman gene expression assays from ABI/ThermoFisher Scientific, Waltham, MA, USA).

    3. Analysis of anti-CD40-siRNAChol-directed cleavage products by 5’RACE.

      Requirements: Gloves, total RNA (from section C1), thermocycler, cDNA synthesis kit, 5’RACE kit.

      1. Synthesize first strand cDNA from 5 μg total RNA (kidney) using the High-Capacity cDNA RT Kit with the gene-specific primer #1 (GSP1): 5’-GCCGACTGGGCAGGGATGACAGACG.

      2. Ligate the adaptor oligonucleotide from the 5’RACE kit to the 5’ end of the cDNA.

      3. Specifically amplify cleavage products using a primer complementary to the adaptor (5’RACE kit) and the nested gene-specific primer #2 (GSP2): GSP2: 5’-AGCCAGGGATACAGGGCGTGTGC. Use the following PCR program: 1 m, 94°C × 60 s and 35 cycles of 94°C × 30 s, 55°C × 30 s and 72°C × 60 s, followed by a final extension of 72°C × 5 min

      4. Check for the presence of a 310 bp amplification product, corresponding to CD40 mRNA specifically cleaved by the anti-CD40 siRNA.

        Use 20 μl PCR product for analysis on a 1% ethidium bromide-stained agarose gel by electrophoresis with a corresponding DNA molecular weight marker.


  3. Histological evaluation of renal lesions in siRNA-treated animals.

    In this section, we will prepare kidney samples for standard histological analysis (embedded in paraffin), immunofluorescence analysis (in OCT), and protein and RNA analysis (stored in RNA stabilization solution).

    Requirements: One of the kidneys obtained in section D2, gloves, ice-cold 1× PBS, tissue molds and cassettes, forceps, paraffin, paraffin dispenser, cold plate, PS buffer (see Recipes), 15% sucrose, 4% paraformaldehyde (PFA), OCT, dry ice, RNA stabilization reagent, high-density polyethylene (HDPE), 50% ethanol, 70% ethanol, 96% ethanol, absolute ethanol, deionized water (MilliQ or similar), microtome.

    1. For standard histological analysis.

      Remove the upper third of a kidney to ensure that both cortical and juxtamedullary glomeruli are present and fix the piece in 5 ml PS buffer at 4°C for 24-48 h. Place the tissue in a labeled cassette (use a pencil, as solvents will dissolve the ink) and prepare for paraffin embedding.

      1. Dip cassettes in a wide-mouth high-density polyethylene (HDPE) 1 L jar filled with 15% sucrose for 24 h before embedding in paraffin blocks.

      2. Dip in 50% ethanol for 2-3 days.

      3. Dip in 70% ethanol for a minimum of 3 h (but can be maintained for up to 7 days).

      4. Dip in 96% ethanol overnight.

      5. Dip in absolute ethanol for a minimum of 3 h.

      6. Wash with clearing agent (xylene) for 1 h at room temperature.

      7. Perform a first paraffin wax at 60°C overnight.

      8. Perform a second paraffin wax at 60°C for a minimum of 3 h.

      9. Place a small amount of molten paraffin in the mold (dispense from a paraffin reservoir). Use warm forceps, transfer the tissue into the mold cut side down, as it was placed in the cassette.

      10. Transfer the mold to a cold plate and gently press the tissue flat. Paraffin will solidify in a thin layer that holds the tissue position.

      11. When the tissue is in the desired orientation, add the labeled tissue cassette to the top of the mold as a backing. Press firmly.

      12. Hot paraffin is added to the mold from the paraffin dispenser. Be sure that there is enough paraffin to cover the face of the plastic cassette. If necessary, fill the cassette with paraffin while cooling, keeping the mold full until solid.

      13. Paraffin solidifies in 30 min. The paraffin block can then be removed and sectioned. If the wax cracks or the tissues are not well aligned, melt them again and start over. Tissue blocks can be stored at room temperature for years.

      14. Tissues are sectioned using a microtome (Leica RM 2155).

        1. Turn on the water bath and check that the temperature is 45°C. Use 1 L fresh deionized water and add 1 g jelly from porcine skin (G1890, Sigma-Aldrich, Sant Louis, MO, USA). Place a fresh blade on the microtome (blades may be used to section up to 10 blocks, but replace if sectioning becomes problematic). The blade should be angled at 5°. Blocks to be sectioned are placed face down for about 15 min on an ice block (cold wax allows thinner sections). After cutting, use forceps to pick up the ribbons of sections and float them on the surface of the water in the water bath to allow for expansion of the sample.

        2. Insert the block into the microtome with the wax block facing the blade, and align the vertical plane. Set the dial to cut 10-μm sections in order to plane the block; once cutting smoothly, set to 2–3 μm-thick sections. Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon.

        3. If the block is ribboning well, cut another four sections, pick them up with forceps or a fine paint brush, and float them on the surface of the water in the 45°C water bath. Float the sections onto the surface of clean glass slides.

        4. If the block is not ribboning well, place it back on the ice block to cool for a longer period to harden the wax.

        5. If the specimens are fragmented when placed in the water bath, then it may be too hot.

        6. Place the slides on the warming block in a 60°C oven for 10 min (so the wax starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature.

    2. For immunofluorescence analysis.

      Place the other pole of the kidney into a tissue mold, coat in OCT, and place it onto dry ice to freeze. Store at -80°C.

      1. Place a small amount of molten OCT (tissue freezing medium, Leica Ref = 14020108926, Leica Biosystems, Richmond, IL) in the mold.

      2. Using forceps, transfer the tissue to the mold, cut side down.

      3. Pour liquid N2 into a polystyrene box containing a 50-ml tube rack (do not submerge the tube rack in the liquid N2). Place the OCT mold on the tube rack and freeze using the vapor.

      4. Wrap with foil and snap freeze in liquid N2 until storage at -80°C.

    1. For protein and RNA extraction.

      Place 3 × 2 mm3 pieces of kidney cortex into 0.5-ml plastic tubes and snap freeze in liquid N2. Store at -80°C. For long-term tissue storage for RNA extraction, place the tissue in five volumes of RNA stabilization reagent and store at -80°C.


  4. Direct immunofluorescence analysis of IgG and C3 deposits in the kidney.

    Requirements: OCT-embedded kidney samples, gloves, ice-cold 1× PBS, cryostat, poly-L-lysine-coated slides (Merck KGaA, catalog number: P0425) or FLEX IHC microscope slides (Agilent, Ref. K8020, Santa Clara, CA, USA), 4% PFA (see Recipes), acetone, blocking solution, conjugated primary antibodies (FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-mouse C3), UltraCruz Tm mounting medium, and DRAQ5.

    1. Prepare the kidney slides for immunofluorescence analysis.

      1. Place the OCT compound mold containing frozen kidney (section D3) at -20°C for 2 h prior to sectioning. Ensure that the [cut] surface of the kidney is carefully placed against the bottom of the OCT mold to enable well-oriented tissue sections.

      2. Use a cryostat to cut 5 µm-thick sections and place them on the surface of poly-L-lysine-coated slides or FLEX IHC microscope slides. Ensure that there are no folds or holes in the tissue section, which can distort tissue morphology. As positive IF controls, use samples of human parotid glands or ganglions; and as negative controls, omit the primary antibodies from the staining process. For the isotypic control, use an irrelevant immunoglobulin of the same isotype, species, and concentration as the primary antibody.

      3. Upon removal from the cryostat, keep at room temperature for 20 min and then fix the slides by submerging into ice-cold pure acetone for 20 min.

      4. Wash the slides 3 times with 100 µl PBS for 10 min each.

      5. Wash the slides twice with distilled water for 5 min each.

      6. Incubate the slides in blocking solution (20% normal goat serum in 1× PBST + 0.2% jelly from porcine skin) overnight at 4°C.

      7. Wash the slides 3 times with 100 µl PBS for 10 min each.

      8. Incubate the sections with primary antibodies (FITC-conjugated goat anti-mouse IgG at 1:300, and FITC-conjugated goat anti-mouse C3 at 1:50) in PBST-jelly buffer containing 1% normal serum. Incubate the slides for 1 h at room temperature in a humidified chamber to avoid the tissue drying out, which would lead to non-specific binding and high background staining.

      9. Wash the slides 3 times in PBS for 5 min each. Primary antibodies can be saved for subsequent experiments.

      10. Mount the samples with a drop of mounting medium containing 1 µg/ml DRAQ5.


  5. Analysis of CD40 expression in the kidney by immunostaining with horseradish peroxidase (HRP).

    Requirements: Gloves, paraffin blocks, pressure cooker, timer, 1× PBS, 0.01 M citrate buffer pH 6, 1% Triton X-100 in PBS, 0.1% fish jelly, bovine serum albumin (BSA), xylene, absolute ethanol, primary antibodies (optimal dilutions and incubation times should be determined for each primary antibody prior to use), biotinylated secondary antibody (135 μl normal serum + 45 μl biotinylated secondary antibody from the ABC staining kit in 10 ml PBS), humidified chamber, ABC (avidin-biotin complex) peroxidase standard staining kit (prepare reagent 30 min before use), DPX mounting medium, DAB substrate.

    Controls: Prepare a negative control with diluent alone (without antibodies) and a positive control with a tissue known to contain the antigen of interest.

    1. Prepare kidney slides for HRP analysis.

      1. Deparaffinize the samples from section D5 by performing standard xylol-ethanol washes.

      2. Wash the slides 3 times in 1% PBS for 5 min each.

      3. Block endogenous peroxidase activity with a methanol wash (30% methanol in PBS + 1% peroxide hydrogen) for 10 min.

        Note: Place the slides on a flat surface. Do not allow the slides to touch each other. Do not allow the sections to dry out.

      4. Wash three times in 1% PBS for 5 min each.

      5. Facilitate antigen retrieval by heating the samples for 5 min in a pressure cooker (on a plastic rack for slides) containing 10 mM citrate buffer, pH 6. This will break protein cross-links after formalin fixation.

      6. Allow to cool slowly at room temperature for 30 min.

      7. Wash twice in 1% Triton X-100 in PBS (PBST) for 5 min each.

      8. To minimize cross-reactivity and reduce non-specific binding caused by hydrophobic interactions, pre-incubate with 20% normal goat (NGS) or horse serum in PBS-Triton + 0.2% jelly at 4°C for 2 h. Remove excess fluid from the slides using a brisk motion and carefully wipe each slide around the sections.

      9. Incubate with primary antibody (anti-CD40 at 1/100, anti-DC-SIGN at 1/50; anti-NF-κB at 1/1,000) in 1% normal goat serum. Apply 100 µl to each slide, covering the tissue sections; tilt each slide from side to side. Incubate in a humidity chamber overnight at 4°C.

      10. Keep at room temperature for 30 min.

      11. Wash 3 times in 1× PBST for 5 min each.

      12. Add 100 µl biotinylated secondary antibodies (at 1/200 + 1% NGS from the Vectastain ABC kit in PBST-jelly). Incubate in the humidified chamber for at least 45 min at room temperature. During this step, it is recommended to prepare the substrate mixture (reagent A = avidin + Reagent B = biotinylated HRP).

      13. Wash 3 times in PBST for 5 min each. Wash well to remove traces of sodium azide as this will inhibit peroxidase activity when developing.

      14. Add the substrate mixture at 1/100 and incubate for 45 min.

      15. Wash 3 times in PBST for 5 min each.

      16. Wash 3 times in PBS for 5 min each.

      17. Incubate the tissue sections with 50 µl Vector DAB substrate in the dark for 3 min or until the desired color reaction is observed when monitored under the microscope. Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle. Discharge DAB in the corresponding recipient. All plasticware that come into contact with DAB must be treated with diluted bleach.

      18. Wash in running tap water for 5 min.

      19. Wash in distilled water.

    2. Counterstain with hematoxylin:

      1. Add hematoxylin (1 vol. hematoxylin solution + 2 vol. distilled water).

      2. Wash with tap water for 5 min.

      3. Wash in distilled water.

      4. Wash with 70% ethanol for 5 min.

      5. Perform three washes with 96% ethanol for 5 min each.

      6. Perform three washes with absolute ethanol for 5 min each.

      7. Perform three washes with xylene for 5 min each.

      8. Mount with DPX.

    3. Microscope examination:

      Sections should be independently examined by two blinded pathologists, and at least 10 fields from each kidney section should be studied at high magnification.

      1. Determine the extent of renal damage. Assess typical glomerular active lesions of lupus nephritis: mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation, and interstitial infiltrates; as well as tubule-interstitial chronic lesions: tubular atrophy and interstitial fibrosis using a Zeiss SteREOLumar V12 microscope from Carl Zeiss AG (Oberkochen, Germany). Grade the lesions semi-quantitatively using a scoring system from 0 to 3 (0 = no changes, 1 = mild, 2 = moderate, and 3 = severe changes).

      2. Quantitate the number of positive cells for each of the markers studied using semi-quantitative evaluation of expression from 0 to 4 (0 = no staining, 1 = staining in <25% of the sample, 2 = staining in 25-50%, 3 = staining in 50–75%, and 4 = staining in 75-100%) in the different compartments of the kidney (glomeruli, vessels, and interstitium). NFκB p65 immunostaining was considered positive when located inside the nuclei.


  6. Evaluation of atherosclerotic lesions in mouse aortas.

    Requirements: ApoE-/- mice, isoflurane chamber, dissection tools (scissors: straight blunt-ended scissors, straight sharp fine scissors, and micro-dissecting spring scissors; tissue forceps: straight serrated-tip forceps, straight or curved serrated-tip fine forceps, and straight fine-tip forceps), syringes, 23 G and 27 G needles, tubes (one EDTA-coated tube, a serum tube, and two lithium heparin-coated tubes), ice-cold PBS, deionized water, RNAzap, 70% ethanol, ice, liquid N2, microscope, cork bed, pinning bed, needles, scalpel, Oil Red O, minutien pins 0.1-mm, Petri dishes, 6× magnification microscope.

    1. Check the extension of ATH lesions in the entire aorta length.

      1. Euthanize mice with isoflurane.

      2. Spray each mouse with 70% ethanol to avoid contamination of the samples.

      3. Excise whole aortas (as modified from Centa et al., 2019) and prepare samples for microscope analysis:

        1. Make a midline incision with scissors from the jugular notch to the pubic bone.

        2. Exsanguinate the mouse by cardiac puncture through the thorax wall (use a 23 G needle). This procedure usually yields 500 μl blood from a 20-week-old mouse. Collect 4 tubes (one EDTA-coated tube, a serum tube, and two lithium heparin-coated tubes). Keep them at room temperature.

        3. Open the abdominal cavity. Cut the parietal peritoneum with scissors in the midline and laterally on both sides.

        4. Open the diaphragm and the chest cavity (cut the rib cage as laterally as possible).

        5. Make an incision in the right auricle for perfusion fluid drainage. Insert a 27 G needle through the apex of the heart in the cranial direction. Keep the needle fixed in the left ventricle while slowly perfusing with 10 ml ice-cold PBS over a minimum of 2 min.

        6. Dissect the liver, spleen, and kidneys.

        7. Cut the trachea and oesophagus on the right side of the heart without damaging the aortic arch. Cut the diaphragm and structures attaching the viscera to the retroperitoneum. Leave the heart, aorta, and kidneys in situ. Fold away the lungs and viscera caudally and cover with a napkin to begin retroperitoneal microdissection of the abdominal aorta (perform microdissection under a microscope at 6× magnification).

        8. Dissect the aortic bifurcation (lift the surrounding tissue with forceps and cut under tension with scissors). Dissect the abdominal aorta cranially. Cut the abdominal branches from the aorta and free the aorta proximally through the aortic hiatus in the diaphragm.

        9. Remove the adipose tissue covering the thoracic aorta. Dissect dorsally of the thymus (carefully) to free the aortic arch with branches. Continue dissecting the carotid arteries as distally as possible in the thoracic cavity. Neck dissection should include carotid bifurcation.

        10. Clean the instruments by sequential rinses in deionized water, RNase decontamination solution, 70% ethanol, and PBS before cutting the aorta.

        11. Lift the heart by the apex with the forceps. Cut the aorta close to the heart and place the whole heart in a tube with PBS. The heart may be stored on ice for a couple of hours before continuing with processing and cryo-mounting of the aortic root.

        12. Cut the aortic arch and place half in a tube containing 1 ml 4% formaldehyde overnight at 4°C.

        13. Dissect the remaining descending aorta, place in a tube, and snap freeze in liquid N2 for RNA analysis.

        14. Place the heart on a cork bed with the ventral side facing up. Fix the heart to the cork with a needle through the apex. Hold the base of the heart with anatomical forceps. Cut away the apical 2/3 of the heart between the two auricles with a scalpel (angled 20° caudally in the sagittal plane and 20° cranially in the transversal plane).

        15. Embed the aortic root in OCT compound (see section F). Store the specimens in zip lock bags at 80°C until cryo-sectioning.

    1. “En face” analysis of the aortic arch and braquiocephalic artery.

      1. Clean the aorta from the remaining periadventitial adipose tissue under a microscope (since Oil Red O stains most hydrophobic and neutral lipids such as triglycerides, diacylglycerols, and cholesterol esters, it is crucial to remove all such tissue at this point). Use scissors and forceps without manipulating or damaging the aorta. Always keep the aorta moist by applying additional PBS when needed.

      2. Place the cleaned and fixed aorta in a 1.5-ml tube (one aorta per tube).

      3. Add 1 ml 78% methanol to each tube and place on a tilted roller with gentle movement for 5 min. Replace the methanol solution and repeat this step twice.

      4. Discard the methanol and add 1 ml fresh Oil Red O.

      5. Incubate the tube on the tilted roller for 50-60 min.

      6. Transfer the aorta to a clean tube and wash twice with 1 ml 78% methanol for 5 min each on the tilter roller.

      7. Discard the methanol and refill the tube with 1 ml PBS.

        Note: If necessary, at this step, aortas can be stored at 4°C.

      8. Prepare a pinning bed: Place a sheet of 25 × 25 mm paraffin-wax film, wrapped with black electrical insulation tape, on a cork bed to make a dark background for the aorta. Place a label on the backside of the pinning bed and use a lead pencil to write the mouse identification number (normal pen ink will disappear in the staining process).

      9. Transfer the fixed aortic arch to the pinning bed and place a drop of PBS on top.

      10. Remove any small stained remnants of adventitial fat (carefully by microscope examination).

      11. Cut open the aorta longitudinally to expose the intimal surface. Introduce the tips of a pair of micro-dissection spring scissors into the artery lumen and cut the outer curvature of the aortic arch from the ascending arch to the left subclavian artery. Begin to cut the outer curvature of the ascending arc in the distal direction and continue to cut open the branches, including the brachiocephalic artery. Continue to cut along the length of the thoracic aorta.

      12. Cut open the lesser curvature and fold open the aorta to display the intimal surface.

      13. Pin the open arch to the pinning bed using the blunt end of minutien insect pins. Gently bend the pins away from the specimen when in place. Pin the aorta flat on the bed without stretching the specimen. Store the pinned arch facing downwards in a Petri dish filled with PBS at 4°C.

      14. Acquire images with a Zeiss steREOLumar V12 microscope connected to a RGB camera (ProgRes C F scan). Place a ruler next to the aorta for calibration of the image.

    2. Quantitate the extension of atherosclerotic plaques.

      Use image analysis software (ImageJ) to determine the lesion area and total intima surface. Lesion quantitation should be performed in a blinded fashion, and it is advisable that a second investigator confirms the results.

      1. Calculate the total arch area: In ImageJ, select the “polygon selection” tool and encircle the total arch area by repetitive clicks. Select “measure” in the analyze menu to display the total arch area in the result window.

      2. Calculate the lesion-free area: In ImageJ, select the “freehand selection” tool and encircle all plaques (stained in an orange-red color) in the arch area while pressing the Alt key. Click “measure” in the analyze menu to display the lesion-free area in the result window.

      3. Calculate the relative lesion area by subtracting the lesion-free from the total arch area and dividing the result by the total arch area.

    1. Cryo-sectioning of the aortic root.

      1. Set the cryostat temperature at -20°C and section thickness to 10 μm. Mount the OCT block containing the aortic root on the specimen holder with the ventricular tissue facing outward. While starting to cut, fine-tune the alignment of the section surface to be parallel to the specimen holder.

      2. Remove the surrounding excess OCT to make it easier to collect the sections without folds. The aortic root should be positioned perpendicularly to the knife blade.

      3. Collect initial control sections on ordinary microscope slides and discard. The first sections should only contain heart muscle tissue. Progress the sectioning by 200 μm. Collect a section and check the progress under a light microscope.

      4. When getting closer to the left ventricle outflow tract, check every 100 μm under the microscope. When initial indications of a vessel wall are observed, slow down the pace to 50 μm. When the first aortic valve appears, this will be point zero for collecting sections. It can be difficult to see exactly when the valves appear, but an exact localization is crucial to perform comparations of lesions in the same region.

      5. Tilt the specimen toward the point zero valve to align the section plane with the two other valves. This is crucial for obtaining true cross-sections of the aorta. Make a drawing of the aortic root, indicating the valves as they appear, and count every 10-μm section that is cut from the point zero onward. When a second aortic valve appears, slightly tilt the specimen again from the valve to align the specimen with the third valve. The distance from where the first aortic valve leaflet appeared to where all 3 of the aortic valve leaflets appeared together did not exceed 80 μm. The first 5-μm thick section with the 3 aortic valve leaflets was mounted onto a glass slide. The adjoining 8 sections, including the first, were collected one by one onto 8 glass slides and marked in order from 1 to 8. The 9th section was collected onto the first glass slide, the 10th onto the second glass slide, and so forth, until each of the 8 glass slides held 8 sections. The 8 frozen sections collected on each glass slide covered a distance of 400 μm.

      6. Fix the sections collected for Oil Red O staining in 4% formaldehyde for 10 min and for immunohistochemistry or immunofluorescence with ice-cold pure acetone for 10 min. Dry at room temperature for 30 min. Store the sections at -20°C.

      7. Capture the images directly using an RGB camera (ProgRes C F scan) attached to a light microscope (NIKON E800). Save high resolution images, preferably in tagged image file (TIFF) format.

    1. Morphometric image analysis using ImageJ.

      The lesion quantitation should be performed in a blinded fashion, and it is advisable that a second investigator confirms the obtained results.

      1. Use the area quantitation feature in the image analysis software to define the total vessel area by encircling the external elastic lamina of the aortic vessel. In ImageJ, select the “polygon selection” tool and encircle the area by repetitive clicks. Then select “measure” in the analyze menu. The total vessel area is displayed in the result window.

      2. Continue to quantitate the ATH lesions in the intimal layer of the vessel, defined by the internal elastic lamina and the luminal boundary. Usually, lesions on the aortic valves are excluded from measurement. In ImageJ, select the “freehand selection” tool and encircle all plaques while pressing the Alt key. Select “measure” in the analyze menu to display the lesion-free vessel area in the result window.

      3. Calculate the relative lesion area by subtracting the lesion-free area from the total vessel area and dividing the result by the total vessel area. Calibrate the results in the image analysis software according to the magnification used in order to obtain the absolute lesion area in µm2.

    Data analysis

    The cholesterol-conjugated, specific anti-CD40 siRNA demonstrated efficient transfection efficiency in dendritic cells (DCs) without decreasing cell viability (evaluated by propidium iodide). Here, we demonstrate the inhibition of CD40 expression and the subcellular localization of the anti-CD40-siRNAChol in the cytoplasm of DCs at 45 min post-transfection (Ripoll et al., 2013). The specificity of siRNA cleavage of CD40 mRNA was confirmed by 5’RACE.

        In the silencing experiments (Ripoll et al., 2013), we firstly matured DCs with LPS (confirmed by an increase in CD40, CD80, and CD86 cell surface expression as measured by flow cytometry) and transfected them with anti-CD40-siRNAChol, which caused a 35% decrease in CD40 expression as compared with the scrambled controls, as well as a significant reduction in the release of TNFα, MCP1, and IL6. In addition, LPS increased CD40 mRNA expression by almost 20-fold (in the kidney) and 35-fold (in the liver) as compared with control levels in a mouse model. CD40 mRNA expression returned to baseline after 24 h in the kidney and after 48 h in the liver. A single anti-CD40-siRNAChol administration reduced renal and hepatic CD40 mRNA expression by 65% and 60%, respectively, for 3 days as compared with control values, with the effects persisting for up to 5 days.

        In the lupus nephritis model (Ripoll et al., 2013), Cy5.5-labeled anti-CD40-siRNAChol was localized in tubular cells from the kidney (Figure 1). In this disease model, we studied the inhibitory effects of anti-CD40-siRNAChol on animal survival, renal function, inflammatory mediators, anti-DNA antibody levels, and renal lesions. For these experiments, mice were distributed into one of five groups subjected to different treatments:

    1. CYP group (n = 9): 50 mg/kg i.p. CYP every 10 days

    2. CTLA4 group (n = 9): 50 µg i.p CTLA4 (ORENCIA, Abatacept, Bristol Myers Squibb, Uxbridge, UK) three times a week

    3. Anti-CD40-siRNAChol-1w group (n = 9): 50 µg i.p. anti-CD40-siRNAChol once a week

    4. Anti-CD40-siRNAChol-2w group (n = 8): 50 µg i.p. anti-CD40-siRNAChol twice a week

    5. Control group (n = 15): 50 µg i.p. ss-control siRNAChol twice a week



      Figure 1. In the lupus nephritis model, Cy5.5-labeled anti-CD40-siRNAChol was localized in tubular cells from the kidney. A. Quantitation of renal internalization of Chol-siRNA administered i.v./i.p. in lupus mice. B. Representative kidney photomicrographs (×400) of: 1) basal autofluorescence; 2) i.v. administration; and 3) i.p. administration. Data are expressed as the mean ± SEM of four separate experiments. *P <0.05 vs. basal. doi:10.1371/journal.pone.0065068.g005.


    Animal survival (by the Kaplan-Meier method) was 100% for the CYP, CTLA4, and CD40-2w groups; 88% for the CD40-siRNA-1w group, and 73% for the untreated group at the end of follow-up. Mice treated with siCD40-2w showed no increase in proteinuria or albuminuria and displayed a dose-dependent reduction in total IgG, anti-dsDNA antibodies, and all IgG fractions. The levels of IgM, IgA, and IgE, as well as pro-inflammatory cytokines such IL2, TNF, IFNγ, MCP1, and IL6, were also reduced in the anti-CD40-siRNAChol-2w group. Histological lesions were graded semi-quantitatively using a scoring system from 0 to 3 (Control = 8 ± 1.5; CYP = 4.3 ± 1.1; CTLA4 = 1.6 ± 0.7; anti-CD40-siRNAChol-1w = 3.8 ± 1.5; anti-CD40-siRNAChol-2w = 1.6 ± 0.6), and IgG and C3 glomerular deposits were reduced in all treated groups (Figures 2 and 3). The anti-CD40-siRNAChol-2w group showed an absence of extra-capillary proliferation, interstitial infiltrates, tubular atrophy, and interstitial fibrosis. Infiltrating CD3+ cells in the tubule-interstitial space were significantly reduced in all treatment groups except siCD40-1w. Furthermore, kidney expression levels of CD40, C3 (a manifestation of local complement synthesis), and pro-inflammatory cytokines were reduced in anti-CD40-siRNAChol-2w mice (Figure 4). In addition, a significant reduction in CD40 protein expression was observed in the interstitial, glomerular, and vascular compartments of the kidneys and in the circulation in the antiCD40-siRNAChol-2w group.

        Finally, we used the CD40 silencing strategy to reduce the progression of atherosclerotic (ATH) lesions in ApoE-/- mice (Hueso et al., 2016). We distributed the mice into 8 treatment groups:

  1. Basal B/8w (n = 5)

  2. ss-control siRNAChol/10 w (n = 5)

  3. ss-control siRNAChol/24w (n = 5)

  4. Scrambled oligonucleotide control SC/14w (n = 5)

  5. Anti-CD40-siRNAChol/10w (n = 5)

  6. Anti-CD40-siRNAChol/14w (n = 5)

  7. Anti-CD40-siRNAChol/24w (n = 10)

  8. Vehicle (n = 5)


The “en face” analysis of whole aorta stained with Oil Red O confirmed that the number and extension of ATH plaque areas decreased in anti-CD40-siRNAChol/24w as compared with control values. Less F4/80 infiltrating macrophages were detected in the vessel walls from anti-CD40-siRNAChol/24w animals, suggesting a role for CD40 in the recruitment of macrophages to the plaque. Finally, less NF-κB+ cells were detected in the intima of anti-CD40-siRNAChol/24w mice, indicating that the protective effect of CD40 silencing may be mediated by NF-κB signaling. Since the strategy aimed to systemically silence CD40, a reduction in the splenic populations of CD3+CD40+ (T-lymphocytic) and CD11b+CD40+ (monocytic) cells was observed in the anti-CD40-siRNAChol/24w animals, suggesting that the reduction in atherosclerotic lesions may be associated with anti-inflammatory mechanisms in the vessel wall via systemic effects.



Figure 2. Histopathological lesions in the lupus nephritis model silenced by CD40 siRNA. A. Costimulatory blockade reduced the lesions in lupus nephritis. B. Representative photomicrograph (×200) of the renal histology from each group. Data are expressed as the mean ± SEM. *P < 0.05 vs. untreated, P < 0.01 vs. untreated. doi: 10.1371/journal.pone



Figure 3. Immunohistochemical analysis of renal IgG and C3 in the lupus nephritis model silenced by CD40 siRNA. A. Deposits of renal IgG and C3 were quantitated by confocal microscopy (MFI). All treatments reduced glomerular deposits. B. Representative photomicrographs of C3 deposits (×630) for each group. Data are expressed as the mean ± SEM. *P < 0.05 vs. untreated, P < 0.01 vs. untreated. doi:10.1371/journal.pone.0065068.g006.



Figure 4. Systemic circulating inflammatory cytokines and local CD40 immunostaining. A. Lupus nephritis promoted the overexpression of CD40 in the serum; this immune modulatory protein was reduced in all therapies. Lupus nephritis also induced an increase in other inflammatory cytokines; treatment with anti-CD40 siRNA reduced some of them. B. Immune localization and quantitation of CD40 protein in different kidney compartments; Chol-siRNA reduced CD40 expression, especially in interstitial cells and vessels. Data are expressed as the mean ± SEM. a P < 0.05 vs. untreated; b vs. CTLA4; c vs. CYP; and d vs. siCD40-2w. doi:10.1371/journal.pone.0065068.g007.

Recipes

  1. Annealing buffer 10×

    1 M Tris-HCl (pH 7.5) 5 ml

    5 M NaCl 2 ml

    DEPC-treated water 43 ml

  2. DEPC-treated water

    Diethyl pirocarbonate (DEPC) 0.1% 1 ml

    Distilled water 1,000 ml

    Mix well and leave at room temperature for 1 h

    Autoclave

    Let cool to room temperature prior to use

  3. 1 M Tris-HCl, pH 7.5

    Tris 121.14 g

    Distilled water 800 ml

    Adjust the pH to 7.5 with the appropriate volume of concentrated HCl

    Bring the final volume to 1 L with deionized water

  4. 12% acrylamide gel

    For 15 ml, enough for a 13 cm × 15 cm × 0.75 mm thick gel:

    10× TBE 1.5 ml

    40% acrylamide (acrylamide: bis acrylamide = 29:1) 4.5 ml

    Distilled and deionized water 9 ml

    Stir to mix, then add:

    10% ammonium persulfate 150 μl

    TEMED 15 μl

    Mix briefly after adding the last 2 ingredients and pour the gel immediately

  5. Non-denaturing gel loading buffer

    Sucrose 50%

    Bromophenol Blue 0.25%

    Xylene cyanole 0.25%

  6. 4% PFA solution in PBS

    Caution: Formaldehyde is toxic

    Requirements: Gloves, safety glasses, fume hood

    1. Place PBS 1× 800 ml in a glass beaker on a stir plate in a ventilated hood

    2. Heat to 60°C while stirring (take care that the solution does not boil)

    3. Add 40 g paraformaldehyde power

    4. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears

    5. Cool the solution and filter

    6. Adjust the volume of the solution to 1 L with 1× PBS

    7. Recheck the pH and adjust with small amounts of diluted HCl to 6.9

    8. Solution can be aliquoted and frozen or stored at 2.8°C for up to one month

  7. 0.1 M citrate stock buffer, pH 6

    Distilled water 800 ml

    Citric acid (MW = 192.1 g/mol) 11.341 g

    Sodium citrate (MW = 294.1 g/mol) 12.044 g

    Adjust the solution to the final desired pH using HCl or NaOH

    Add distilled water to 1,000 ml

    Store at room temperature (shelf life up to 3 months)

  8. 0.01 M citrate buffer

    0.1 M citrate buffer 100 ml

    Distilled water to 900 ml

  9. Complete culture medium

    RPMI 1640 medium 500 ml

    100 U/ml penicillin

    100 μg/ml streptomycin

    2 M L-glutamine

    10% heat-inactivated and filtered FBS

    20 ng/ml GM-CSF

  10. 0.3% Oil Red O stock solution

    Oil Red O 0.3 g

    Isopropanol 99% 100 ml

    Dissolve the solution at 56°C for 1 h

    Filter the solution through Whatman No. 1 filter paper and keep at 4°C

    This solution is stable for 1 year

  11. Oil Red O working solution

    Prepare in a fume hood

    Stock solution 60 ml

    Distilled water 40 ml

    Filter the solution through Whatman No. 1 filter paper

    The solution is stable for no longer than 2 h and must be prepared 15 min before use

  12. Phosphate-buffered saline (PBS), pH 7.5

    0.1 M phosphate

    0.15 M NaCl

  13. PBS-Triton (PBST)

    Triton-X 2 ml

    PBS 1,000 ml

    PBST/Jelly

    Jelly from porcine skin 0.1 g

    PBST 50 ml

  14. PS Buffer

    Formalin 10% 200 ml

    Water, quality MilliQ or similar 200 ml

    Heat 60-70°C

    Add 1 pellet NaOH

    Cool in water to 24°C

    Add 37.5 g D+ sucrose

    Add 2 ml 0.5 M EDTA and 2.5 ml BHT (butylated hidroxytoluene)

    Adjust to pH 7.4 with 1 M NaOH

    Add MilliQ water to 500 ml and filter using filter paper with a medium filtration rate particle retention of 10-20 µm

  15. Secondary antibodies (for a 1:200 dilution)

    Antibody 0.5 μl

    Serum normal goat (or horse) 20% 1 μl

    PBST/Jelly 98.5 μl

  16. Serum normal goat or horse 20%

    Serum from normal goat or horse 20 μl

    PBST/Jelly 80 μl

Acknowledgments

This study was partially funded by Instituto de Salud Carlos III (Co-funded by the European Regional Development Fund. ERDF, a way to build Europe) through the projects PI11/00556, PI14/00762, and PI18/01108 and by REDinREN (12/0021). We thank REDinREN and the CERCA program/Generalitat de Catalunya for institutional support.

Competing interests

There are no conflicts of interest.

Ethics

The experiments were carried out in accordance with EU legislation on animal experimentation and were approved by CEEA: Animal Experimentation Ethics Committee, the Institutional Ethics UB Committee for Animal Research.

References

  1. Arranz, A., Reinsch, C., Papadakis, K. A., Dieckmann, A., Rauchhaus, U., Androulidaki, A., Zacharioudaki, V., Margioris, A. N., Tsatsanis, C. and Panzner, S. (2013). Treatment of experimental murine colitis with CD40 antisense oligonucleotides delivered in amphoteric liposomes. J Control Release 165(3): 163-172.
  2. Bohula, E. A., Salisbury, A. J., Sohail, M., Playford, M. P., Riedemann, J., Southern, E. M. and Macaulay, V. M. (2003). The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem 278(18): 15991-15997.
  3. Bosmans, L. A., Bosch, L., Kusters, P. J. H., Lutgens, E. and Seijkens, T. T. P. (2020). The CD40-CD40L Dyad as Immunotherapeutic Target in Cardiovascular Disease. J Cardiovasc Transl Res 3..
  4. Centa, M., Ketelhuth, D. F. J., Malin, S. and Gistera, A. (2019). Quantification of Atherosclerosis in Mice. J Vis Exp(148).
  5. de Ramon, L., Ripoll, E., Merino, A., Lucia, M., Aran, J. M., Perez-Rentero, S., Lloberas, N., Cruzado, J. M., Grinyo, J. M. and Torras, J. (2015). CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury. Kidney Int 88(3): 538-549.
  6. Elgueta, R., Benson, M. J., de Vries, V. C., Wasiuk, A., Guo, Y. and Noelle, R. J. (2009). Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 229(1): 152-172.
  7. Hueso, M., de Ramon, L., Navarro, E., Ripoll, E., Cruzado, J. M., Grinyo, J. M. and Torras, J. (2016) Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis. Atherosclerosis 255:80-89.
  8. Karnell, J. L., Rieder, S. A., Ettinger, R. and Kolbeck, R. (2019). Targeting the CD40-CD40L pathway in autoimmune diseases: Humoral immunity and beyond. Adv Drug Deliv Rev 141: 92-103.
  9. Kretschmer-Kazemi Far, R. and Sczakiel, G. (2003). The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res 31(15): 4417-4424.
  10. Lutgens, E., Lievens, D., Beckers, L., Wijnands, E., Soehnlein, O., Zernecke, A., Seijkens, T., Engel, D., Cleutjens, J., Keller, A. M., Naik, S. H., Boon, L., Oufella, H. A., Mallat, Z., Ahonen, C. L., Noelle, R. J., de Winther, M. P., Daemen, M. J., Biessen, E. A. and Weber, C. (2010). Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile. J Exp Med 207(2): 391-404.
  11. Parasuraman, S., Kumar, E., Kumar, A. and Emerson, S. (2010). Free radical scavenging property and diuretic effect of triglize, a polyherbal formulation in experimental models. J Pharmacol Pharmacother 1(1): 38-41.
  12. Pluvinet, R., Petriz, J., Torras, J., Herrero-Fresneda, I., Cruzado, J. M., Grinyo, J. M. and Aran, J. M. (2005) RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells. Blood 104(12): 3642-3646.
  13. Remer, M., White, A., Glennie, M., Al-Shamkhani, A. and Johnson, P. (2017). The Use of Anti-CD40 mAb in Cancer. Curr Top MicrobiolImmunol 405: 165-207.
  14. Ripoll, E., Merino, A., Herrero-Fresneda, I., Aran, J. M., Goma, M., Bolanos, N., de Ramon, L., Bestard, O., Cruzado, J. M., Grinyo, J. M. and Torras, J. (2013). CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms. PLoS One 8(6): e65068.
  15. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R. and Saigo, K. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32(3): 936-948.
  16. Hueso, M., Casas, A., Mallén, A, de Ramón, L., Bolaños, N., Varela, C,. Cruzado, JM., Torras, J., Navarro, E. (2019). The double edge of anti-CD40 siRNA therapy: It increases renal microcapillar density but favours the generation of an inflammatory milieu in the kidneys of ApoE−/− mice. J Inflamm (Lond) 16: 25-34.

简介

[摘要]所述的共刺激分子CD40和其配体CD40L发挥在免疫过程的调控中起关键作用,并参与自身免疫和炎性疾病的病理生理学。抑制CD40 - CD40L轴是一种有前途的疗法,并且已经设计了许多策略和技术来阻碍其功能。我们集团有使用沉默CD40的广泛经验RNAi技术,在这里,我们总结一下协议的具体抗CD40 siRNA在不同的全身用药啮齿动物模型,除了后续孔定量FIC通过免疫染色CD40表达的小鼠肾脏通货膨胀。使用RNAi技术具有特定的siRNA沉默基因的正成为一个重要的方法来investigat È基因功能和正在迅速成为治疗工具。


图形摘要:


CD40 siRNA机制

[背景]共刺激分子CD40及其配体CD40L是涉及自身免疫性和炎症性疾病(包括癌症,移植物抗宿主病,炎症性肠病,系统性红斑狼疮)的病理生理学的最著名的免疫检查点之一。红斑狼疮(SLE) ,类风湿性关节炎,类1型糖尿病,移植排斥反应,和动脉粥样硬化(Lutgens等人; 2010; Ripoll会面。等人,2013 ; deRamón等人。,2015; Hueso等人。,2016; Hueso等。 ,2019; Karnell等,2019)。CD40是43 - 50 kDa的跨膜蛋白属于肿瘤坏死因子(TNF)受体超家族和许多免疫细胞的表面上表达。CD40与其配体CD40L(CD154)相互作用诱导CD40并刺激的下游信号传导的三聚,包括NF- κ即上调乙途径的促炎基因(Elgueta等人,2009)。因此,抑制CD40 - CD40L二联体是一种有前途的疗法,并且已设计出许多策略和技术来阻碍其功能,例如施用CD40与TRAF6相互作用的小分子抑制剂(Bosmans等, 2020 ),脂质体装载抗CD40反义寡核苷酸(ASO)(ARRANZ等人,2013),抗CD40的siRNA(Pluvinet等人,2004; deRamón等人。,2015 ; Hueso等人。,2016) ,或单克隆抗体(Remer et al。,2017)。目前,有3次临床试验测试抗CD40单克隆抗体,CFZ533的安全性和有效性,防止急性排斥反应肾(NCT03663335)或肝(NCT03781414)移植的患者,并评价其效果Ø n个肾脏的患者升upus ñ ephritis(NCT03610516)。其他正在开发的临床试验中使用的抗CD40L抗体(NCT03605927)预防急性移植物抗宿主病(NCT03605927) ,或溶瘤腺病毒载体是表达抗CD40抗体(NCT03852511)治疗晚期或转移性肿瘤。我们小组已经开发化学上稳定,胆固醇-缀合的小抑制RNA抗鼠CD40分子和报道其效力,分布的评价,并影响耐久性以下SYSTE MIC给药(Ripoll会面等人,2013; deRamón等人,2015。 ,Hueso等人,2016,Hueso等人,2019)。她即我们概括描述这一特异性抗CD40 siRNA的全身用药在不同的M协议乌斯车型。

关键字:siRNA, CD40, CD40L, 胆固醇衍生的寡核苷酸, 肾脏, siRNA治疗, 非目标效应



材料和[R eagents


消耗品
d isposable手套
无RNAase的1.5 ml聚丙烯试管
96孔板
0.5 ml聚丙烯管
50毫升猎鹰锥形管小号(康宁,目录号:45352054)
70 μ米尼龙细胞滤网小号(BD Biosciences公司,目录号:45352350)
细胞培养板
针筒
毛巾
样品收集管
23-25 G针
2 ml无RNAase的Eppendorf管(Merck KGaA ,目录号:T2795)
P OLY -L-赖氨酸-包被的载玻片(默克KGaA公司,CATA登录号:P0425)
Whatman 1号滤纸


试剂和ķ其
无核酸酶的水(经DEPC处理的水)
绝对异丙醇(Merck KGaA ,目录号:I9516)
Tris碱(Tris-羟甲基-氨基甲烷; Bio-Rad Lab,目录号:161-0719)
3 M醋酸钠(Merck KGaA ,目录号:S7899)
溴酚蓝二甲苯氰染料溶液(Merck KGaA ,目录号:B3269-5mL)
N,N,N”,N'-吨etramethylethylenediamine ,TEMED(BIO-R广告,赫拉克勒斯,目录号:161-0800)
Amonium p ersulfate,APS (BIO-R广告,赫拉克勒斯,目录号:161-0700)
甲醇(Merck KGaA ,目录号:82762)
绝对乙醇(从常规化学药品供应商处获得)
过氧化氢(从一般化学品供应商处获得)
D +蔗糖(AppliChem GmbH,目录号:200-334-9)
乙二胺四乙酸(EDTA,Merck KGaA,目录号:E6758)
RNase-ZAP(Merck KGaA ,目录号:R2020)
BHT(丁羟甲苯;默克(Merck)KGaA ,目录号:W218405)
FACS升ysing小号olution(Becton Dickinson公司,目录号:349202)
二甲苯(异构体混合物,VWR International,目录号:28975.360)
DPX安装介质(VWR International,目录号:360294H)
来自猪皮的果冻(Sigma-Aldrich,目录号:G1890)
油红色O (Merck KGaA,目录号:O0625)
RPMI 1640培养基(生物工业)
注:- [R eagents 1-2 0被小号在室温下tored 。


Opti-MEM(Themo Fisher,目录号:51985034)
胎b绵羊小号erum(龙沙制药和生物技术,目录号:14-802F)
白蛋白ELISA试剂盒(Active Motif)
DEPC(焦碳酸二乙酯; Merck KGaA ,目录号:D5758)。
40%丙烯酰胺/ Bis 29:1(Bio-R ad ,Hercules,目录号:161-0146)
碘化丙我odine(干细胞技术,目录号:75002)
TRIzol (ThermoFisher Scientific,目录号:15596026)
DAB底物(3,3'二氨基联苯胺四盐酸盐水合物,Sigma-Aldrich,目录号:D5637-5G)
流Ç ytometry小号泰宁b uffer(赛默飞世科学,目录号:00-4222)
100 mM青霉素/链霉素(ThermoFisher Scientific,目录号:15070-063)
注:- [R eagents 2 1 - 3级0是小号在4 tored ℃下。


200 mM L-谷氨酰胺(ThermoFisher Scientific,目录号:25030-024)
高容量cDNA反转录试剂盒(ThermoFisher Scientific,目录号:4368814)
PureLinkTMRNA Mini试剂盒(ThermoFisher Scientific,目录号:12183020)
用于快速扩增cDNA末端试剂盒的5'RACE系统(ThermoFisher Scientific)
Oligofectamine 2000(ThermoFisher Scientific,目录号:12252011)
GM-CSF(研发系统,目录号:215-GM)
从脂多糖(LPS)E. Ç OLI血清型O111:B4(Sigma-Aldrich公司,目录号:L4391)
GeneEraser荧光素酶抑制测试系统(Stratagene / Agilent ,目录号:240192)
萤光素酶测定系统(Promega,目录号:E1531)
Polyfect吨ransfection ř eagent(Qiagen公司,目录号:301105 )
血清正常山羊(Merck KGaA ,目录号:G9023)或马20%(Merck KGaA ,目录号:H0146)
注:- [R eagents 3 1 - 41储存在-20 ℃下。


的Taqman基因表达测定(ABI /赛默飞科学)(Ť能够1)


表1.本研究中使用的商业TaqMan探针


(1)使用的Taqman探针的商业代码(ThermoFisher Scientific)。


(2)Refseq / Genbank所测试基因的唯一标识符。


(3)探针内的基本位置。


0.1 M柠檬酸盐缓冲液,pH 6 (请参见食谱),在室温下保存
0.01 M柠檬酸盐缓冲液(请参阅食谱),在室温下保存
油红色工作溶液(请参见食谱),在室温下保存
Phosph一个TE缓冲小号艾琳(PBS),pH为7.5(参见食谱),贮存在室温下
PBS-Triton(PBST)(请参阅食谱),在室温下保存
PS b uffer (见配方),贮存在室温下
完整Ç ulture米edium (见配方),保存于4 ℃下
二抗(对于1:200稀释度)(请参见食谱),保持在-20 °C
退火b uffer 10 ×(见配方)
经DEPC处理的水(请参阅配方)
1 M Tris-HCl,pH 7.5 (请参阅食谱)
12%丙烯酰胺凝胶(请参阅食谱)
非-变性凝胶样缓冲液(见食谱)
PBS中的4%PFA溶液(请参阅食谱)
0.3%的油红O小号滴答小号olution (见食谱)
正常山羊或马的血清浓度为20%(请参阅食谱)


小学一ntibodies
在这项工作中,我们使用了以下特定的第一抗体:


兔多克隆抗CD40抗体(C-20,圣克鲁斯生物技术,目录号:sc-975)
兔多抗CD154抗体(H215,圣克鲁斯生物技术,目录号:sc-9097)
小鼠单克隆抗DC-SIGN抗体(1B10,圣克鲁斯生物技术,目录号:sc-23926)
兔多克隆抗NF- κ乙p65的抗体(抗-磷酸S536,Abcam公司,目录号:Ab86299)
抗F4 / 80抗体(Hycult Biotech,目录号:HM1066)
ř abbit抗小鼠胶原IV抗体(Chemicon公司国际,目录号:AB756p)
ģ燕麦多克隆抗-人体C3c抗体缀合到FITC(Nordic- MuBio ,目录号:GAHu /体C3c / FITC)


此外,以下来自BD Biosciences (美国加利福尼亚州圣何塞)的抗体用于流式细胞仪:


抗CD11c抗体(克隆HL3)
抗CD11b抗体(克隆M1 / 70)
抗CD40抗体(克隆HM40-3)
抗CD80抗体(克隆16-10A1)
抗CD86抗体(克隆GL1)
注意:所有一抗都保存在-20 °C ,除非制造商明确规定了其他温度。


二抗和组织学试剂
在该工作中使用了以下二抗:


Alexa 488标记的鸡肉防山羊(ThermoFisher Scientific,目录号:AB_2535870)
山羊抗兔(ThermoFisher Scientific,目录号:AB_2633280)
Alexa 555标记的山羊抗小鼠(ThermoFisher Scientific,目录号:AB_2535844)
Alexa 546标记的山羊抗兔(ThermoFisher Scientific,目录号:AB_2633280)
未结合的山羊抗大鼠(Novus Biologicals)
生物素化的马抗山羊(Vector Laboratories,目录号:BA-9500)
FITC缀合的山羊抗小鼠(Merck KGaA ,目录号:F0257)
VECTASTAIN Elite ABC HRP试剂盒(Vector实验室,目录号:PK-6100)
抗生物素蛋白/生物素封闭试剂盒(Vector实验室,目录号:PK-4001-NB) 
Ñ OTE :所有第二抗体储存在4 ℃下,除非一个其他温度是专门制造商规定。           



使用了以下组织学试剂:


哈里斯ħ ematoxylin小号olution(Sigma-Aldrich公司,目录号:HHS32-1L)
曙红Y小号olution ,一个lcoholic(Sigma-Aldrich公司,目录号:HT110132-1L)
高碘酸(Sigma-Aldrich,目录号:P0430-25G)
希夫ř eagent(Sigma-Aldrich公司,目录号:3952016-500ML)
PAS染色试剂盒(Sigma-Aldrich,目录号:101646)
油Red-O试剂(Sigma-Aldrich,目录号:O0625-100G)
组织Tec OCT包合物(Sakura FinetekEurope )
UltraCruz的Tm米ounting米edium(圣克鲁斯生物技术,目录号:SC-24941)
DRAQ5(ThermoFisher Scientific,目录号:65-0880-92)
注意小号:


用于核复染。
所有组织学试剂均在室温下保存。


动物
六个月-老NZB / NZW(F1)的雌性小鼠(杰克逊实验室,查尔斯河,威尔明顿,MA,USA)
六至八周大的雄性ICR小鼠(杰克逊实验室,查尔斯河,威尔明顿,美国马萨诸塞州)
八周-旧的ApoE - / -雌性小鼠(杰克逊实验室,查尔斯河,威尔明顿,MA,USA)
注意:将动物圈养在保持恒温的房间内,并允许其自由饮水和标准实验室饮食。为了加速动脉粥样硬化,载脂蛋白E - / -小鼠饲喂W¯¯含有0.2%的胆固醇和提供42%的西部时代饮食的能量为脂肪(TD.88137; Harlan- Tekland ,麦迪逊,WI,USA)。通过吸入异氟烷使动物安乐死。方案经批准Ë thics Ç ommittee为一个nimal [R UB-的esearch Bellvitge ,按照实验室动物实验的欧洲立法进行和实验。


siRNA寡核苷酸
在这项工作中,我们使用了CD40特异性DS-siRNA的同源两者的小鼠和大鼠CD40 mRNA序列(在本文中抗CD40-siRNA转澈)和一个加扰序列(S / S)DS-的siRNA的控制(在此S / S -对照siRNA(Chol )。通过吡咯烷连接子将胆固醇(chol )分子缀合至有义链的3'末端来修饰两者。


所述的siRNA序列如下:


一个NTI-CD40正义链:5'-GUGUGUUACGUGCAGUGACUU-3'


一个NTI-CD40反义链:5'-GUCACUGCACGUAACACACTG-3'


(s / s),对照siRNA有义链:5'-ACUACAAGACUCGUGAGAAU-3'


(s / s),对照siRNA反义链:5'-UGGUCACGAGUCUUGUAGUUUU-3'


为了确定转染效率和器官分布小号的所述的siRNA ,胆固醇缀合和未修饰的抗CD40的siRNA标记与Cy5.5的。


注意:所有寡核苷酸均购自Microsynth AG(瑞士Balgach ),并保存在-20 °C下。


细胞系
高度可转染的人类胚胎肾脏293FT细胞系(ThermoFisher Scientific,目录号:R70007)
主d endritic细胞(直径:从ICR小鼠的骨髓btained)


设备


细胞刮刀(Sarstedt AG&CO,Nümbrecht ,GE,目录号:83.3950)
手术刀
钳子:小号traight ,锯齿-尖镊子; 直线或曲线,锯齿-尖端细镊子; 直细-尖镊子
剪刀:小号traight ,钝剪刀; 笔直,锋利,细的剪刀; 和微型-解剖弹簧剪刀
自动移液管(1 - 10 μ升,20 - 200 μ升)
血细胞计数器
水浴
Jasco V-650分光光度计(美国马里兰州伊斯顿,用于确定siRNA双链体的熔点)
Olympum a尿量分析仪AU400(德国汉堡,用于测定尿蛋白和肌酐浓度s )
TaqMan实时PCR ABI PRISM ® 7700(赛默飞世科学,用于定量PCR实验)
BD FACS坎托II Ç ytometer(BD Biosciences公司,在流式细胞术实验中使用)
Zeiss SteREOLumar V12显微镜(Carl Zeiss AG,用于显微镜实验)
Leica TCS-SL光谱共焦显微镜(Leica Camera AG,用于显微镜实验)
TD-二十○分之二十光度计(特纳设计,用于升uciferase测定)
T-25 ULTRA-TURRAX TM均化器(IKA ® -Werke GMBH&CO )
NanoDrop 2000c S分光光度计(ThermoFisher Scientific)
台式离心机(Eppendorf冷却离心机,模式:5424R)
细胞Ç ulture CO 2我ncubator(NuAire自动流NU 8700)


软件


徕卡ç onfocal小号oftware(徕卡股份公司,韦茨拉尔,德国)
图像分析软件ProgResCapturePro 2.7.7 (JenoptiK AG,耶拿,GE)
ImageJ v1.48(NIH,美国马里兰州贝塞斯达)
ExpressionSuite软件v1.0或更高版本(ABI,ThermoFisher Scientific,美国马萨诸塞州沃尔瑟姆)
SDS的软件v2.4(ABI,ThermoFisher Scientific,美国马萨诸塞州沃尔瑟姆)
FACS DIVA软件(美国加利福尼亚州圣何塞的BD Biosciences公司)


程序小号


siRNA的合成与制备
要求:d isposable手套,加热水浴,离心,自动移液管(1 - 10 μ升,20 - 200 μ升),的Jasco V-650分光光度计,RNA酶-free 1 。5 -毫升聚丙烯管,冰,无核酸酶水,退火缓冲液(10毫摩尔Tris pH为7.5,20mM的NaCl)中,3M乙酸钠(pH 5.2),异丙醇,70%乙醇,ö ligonucleotides。


抗CD40寡核苷酸的设计。
ANALY ž Ë靶mRNA序列(在本文中鼠CD40,NCBI登录X60592.1 )选择一个数字,并具有以下结构,符合序列的5'-GN 17 C-3' 。产生了九个不同的序列以选择最佳的靶位点(Pluvinet等,2004)。一般规则,以改善siRNA沉默的有效性是:1)p在有义链的5'端的G / C的resence ; 2)在反义链的5'末端存在A / U ;3)在反义链的5'末端的前7个碱基中存在至少5个A / U残基;4)序列中不允许有超过9个G / C残基的运行(Ui-Tei等,2004);5)在靶位点处的二级结构应保持到最低限度(Bohula等人,2003;克雷奇默尔-Kazemi的远东和Sczakiel ,2003)。


寡核苷酸退火以产生ds-siRNA。
对于寡核苷酸退火,可通过混合混合等摩尔量的不同抗CD40和对照siRNA的互补有义链和反义链:


寡核苷酸1 /有义链(100 pmol / µl)……………………………… 10 µl           

寡核苷酸2 /反义链(100 pmol /µl)……..………………………10 µl


2 x寡核苷酸退火缓冲液(10 mM Tris,pH 7.5,20 mM NaCl)………………... 50 µl           

无核酸酶的水(最高)…………………………………………..………… 100 µl           

              在水浴中于90 °C加热3分钟,然后将其关闭。
让试管在水浴中缓慢冷却至室温(<60分钟)。最终浓度应为100 pmol / µl ds-siRNA。
将退火的ds-siRNA寡核苷酸储存在4 °C以便立即使用,或储存在-20 °C以便更长的存储时间。
确定ds-siRNA的解链温度(Tm)。
在Jasco V-650分光光度计中,以0.5 °C / min的线性温度梯度加热样品。获得熔融温度作为一阶导数的最大值。未修饰的CD40的ds-siRNA的熔点是79 ℃,和该中的C ^ holesterol衍生CD40 DS-的siRNA(抗CD40-siRNA转澈)是71 ℃下。


ds-siRNA的酒精沉淀。
添加一个0.1体积的3M乙酸钠(pH5.2)和1倍体积的异丙醇,混匀。
保持在冰上进行5分钟并离心,在所述在微量的最高速度10分钟。
小心吸出上清液,用0.5 ml的70%冷乙醇洗涤沉淀,并小心除去所有乙醇。
空气干燥沉淀为没有再在室温下超过15分钟,并重新悬浮的ds-siRNA对不含核酸酶的水在2 - 5 -倍原体积。
定量的siRNA的浓度使用Ñ ANO d ROP或等效。在F伊纳勒浓度应为20 - 50皮摩尔/微升。
ANALY ž Ë所述的siRNA在非-变性12%聚丙烯酰胺凝胶(见ř ecipes)大小和完整性,并储存在-20 ℃下或-70 ℃下。
混合达5个μ升用2寡核苷酸μ升装载缓冲液(见ř ecipes)。
加载在非样品-变性12%聚丙烯酰胺凝胶和受electrophores是在200 - 250 V.
当停止电泳乙romophenol乙略染料前已经迁移的方式向下凝胶三分之二。
染色为2的凝胶-在1 5分钟μ克/米升的溴化乙锭溶液。
将该凝胶浸泡2 -在水中5分钟。
使用紫外线透射仪可视化siRNA。


所述siRNA应该迁移为21 -稍微运行后面22 bp的条带乙romophenol乙略染料前沿。在主要siRNA谱带后面延伸的第二条强度较低的谱带可能很明显,代表一条部分消化的siRNA链。所述underdigested RNA链是27个核苷酸的长度,并且不产生任何非-当特异性作用用于转染细胞。


反的沉默能力的测定- CD40-siRNA转澈使用GeneEraser荧光素酶抑制测试系统
要求:d isposable手套,96孔板,自动移液管(1 - 10 μ升,20 - 200 μ升),细胞刮刀(Sarstedt的AG&CO。,宁布雷希特,GE,目录号:83.3950),离心,冰,0.5 -毫升聚丙烯管,PTARGET-LUC-rCD40,Oligofectamine转,HEK-293细胞,Polyfect吨ransfection ř eagent,70%乙醇,1 ×磷酸盐缓冲盐水(PBS),1 ×裂解试剂,升uciferase一个SSAY ř eagent, TD-20 / 20发光计。


发电机密封Ë的apTarget-LUC-CD40质粒。
对应于部分鼠CD40 cDNA序列平末端506 bp片段(编码序列的41个核苷酸- 547; GenBank登录Acc编号AF241231)是钝-端在克隆到质粒PTARGET-luc的在荧光素酶的3'UTR基因。


用siRNA(100 nM )转染HEK-293细胞。
准备ds-siRNA的100 nM溶液,使用O ligofectamine按照制造商的说明转染融合的HEK-293细胞。
6小时后,转染上述细胞与400ng的PTARGET-LUC-CD40使用Polyfect吨ransfection ř eagent根据制造商的说明。通过共转染500 ng pCMV-bGal标准化转染效率。
将细胞在37 °C ,5%CO 2的潮湿气氛中孵育48小时。
Prepar Ë细胞裂解物进行分析。
除去的从生长培养基中培养的细胞。
冲洗的细胞在1 × PBS中,并除去尽可能多的。
在一个96孔板,分配20微升/孔1 ×裂解试剂(1 ×裂解试剂通过添加4体积水至1个体积制备5 ×来自试剂盒的裂解试剂)。
刮掉的从培养皿的细胞,将溶液转移到离心管中。
沉淀的通过短暂离心细胞碎片,将上清液转移到一个新试管中。
混合20微升细胞溶胞产物与100μl升uciferase一个SSAY ř eagent并测量的在TD-20/20光度计光。


骨髓来源的树突状细胞(DC)萃取,文化,激活,和转染的siRNA在体外
要求:d isposable手套,50 -米升猎鹰锥形管小号(康宁,目录号:45352054),70 - μ米尼龙细胞滤网小号(BD Biosciences公司,目录号:45352350),高压灭菌材料小号(钳子,解剖刀,剪刀),细胞培养罩,冰,小鼠股骨和胫骨,血球,细胞培养板,样品收集管,Oligofectamine转2000,BD FACS升ysing小号olution,完全RPMI 1640培养基(RPMI 1640培养基补充有100U / ml的p enicillin, 100μg/ ml的小号treptomycin,2μML-谷氨酰胺,10%热-失活的FBS ,和20ng / ml的GM-CSF) ,的Opti-MEM培养基,碘化丙啶(原液:1.5毫摩尔)中,siRNA,LPS,流式细胞仪。


安乐死ICR根据小鼠到该机构的指导方针和提取的股骨胫骨和。
将每只小鼠放在无菌罩中的无菌手术垫上。
用70%的乙醇喷洒鼠标,以免污染样品。
浸泡的在RPMI股骨和胫骨1640培养基。
收获的骨髓(BMC)。
注:所有工作都应该进行无菌条件下在细胞培养罩。


切两端股骨机智^ h无菌剪刀。
用无菌培养皿将b转移到RPMI 1640中。
大力冲洗骨骼的内部用1ml冰冷的RPMI 1640培养基中使用1 -毫升吸管。同花顺2 - 3次,直至骨头完全为白色。
冲洗骨髓出到一个70 - μ置于50米的尼龙细胞过滤器-米升猎鹰锥形管。
粉碎通过细胞过滤骨髓使用5 -毫升柱塞,和洗涤用5微米的过滤器升RPMI。
离心的在250个细胞×克8分钟,在4 ℃下,并丢弃上清液。
重悬的细胞沉淀在1米升RBC裂解缓冲液,孵育在室温下5分钟。
中和裂解缓冲液通过加入5ml FBS 。
离心机的细胞以250 ×克在4 8分钟℃下,弃上清,重悬在5米升RPMI 1640培养基。取出等分的细胞悬液进行计数。
确定获得的细胞总数。
一种。稀10 μ升骨骨髓悬浮液在90 μ升1 × BD FACS升ysing小号olution(见上文)。


b 。计数10 μ升细胞悬浮液使用一个血球。


生长5 × 10 6个细胞。
在5 ml完整RPMI 1640培养基(参见上文)中添加细胞,该培养基中添加了10 ng / ml IL-4和20 ng / ml GM-CSF,在37 °C的T25烧瓶中,5%CO 2的潮湿环境中生长。


24小时后,一些细胞粘附在平板上,但其他细胞则悬浮在培养基中。72小时后出现菌落。n个贴壁细胞的棕土逐渐减少小号随时间和在第5天,将悬浮的细胞与树突突起逐渐增加。在第7天,将悬浮的细胞求我n至骨料和突起伸长。


更改的第3天至5培养基。
删除的中介UM小心,以免干扰生长的细胞。
短暂离心(250 ×g ,8分钟),因为DC s松散附着,所以去除了培养基。重悬细胞沉淀在具有10米的相同的烧瓶升新鲜RPMI 1640补充有20纳克/立方米升GM-CSF。
收获BMDC(第8天)
收集非贴壁细胞通过轻轻吹打的培养基,并转移到无菌的50 -米升离心管小号。丢弃该贴壁细胞的是包括巨噬细胞。
松散粘附的BMDC变得容易通过该方法脱落成悬浮液,而巨噬细胞仍然是一个dhered到培养皿中。
计算获得的细胞总数。
在第8天用siRNA转染。
孵育1 × 10 6未成熟的BMDC / ml的2μM未修饰的siRNA-Cy5.5的或澈-siRNA转-Cy5.5的(见小号的M的挠度Ë aterials和试剂部,上述),使用阳离子脂质Oligofectamine转2000中的Opti - 6 MEM培养基-厘米的菜肴。


在第9天用100 ng / ml LPS处理不成熟的BMDC,持续12 h。
收获BMDC 
收集所有的细胞(分离细胞一刮刀)。
离心机中以250细胞×克为8分钟。
除去上清液,将细胞沉淀重悬于5 ml PBS中(洗涤1)
离心机中以250细胞×克为8分钟。
除去上清液并将细胞沉淀重悬于5 ml PBS中(洗涤2)
离心机中以250细胞×克为8分钟。
重悬在1米的细胞沉淀升RPMI 16 4 0补充有10%FBS和20 mM的青霉素/链霉素。
数细胞。
染色的带的终浓度的DCs ≤ 1 μ克/ ml的碘化丙啶碘化物(PI)。
通过将固体PI溶解在PBS中制备1 mg / ml (1.5 M)的储备溶液。避免长时间暴露在光线下。
只要有可能,准备和使用的当天原液。如果在储备溶液必须提前,等分试样并储存在-20制成在密闭小瓶中℃下(通常最多稳定到1个月)。
重悬50 μ升细胞悬浮液在50 μ升˚F低Ç ytometry小号泰宁b uffer。
加入5 μ升PI染色小号每100 olution μ升细胞悬浮液。不洗的细胞加入PI之后。
在黑暗中于冰上孵育5 – 15分钟。
ANALY ž Ë的样品立即通过流式细胞术。
表征的通过流式细胞术细胞表型。
非特异性块Fc介导的与0.5相互作用μ克抗小鼠CD16 /每100 CD32 μ升15分钟,在4 ℃下染色之前。
抗体结合动力学是温度依赖性的。在冰上染色可能需要更长的孵育时间。


等分试样50 μ升细胞悬浮液到每个管(一个每抗体)。
结合的原发性抗体(同种型对照和下列抗体的细胞悬浮液:抗CD11c(HL3克隆),抗CD11b(M1克隆/ 70),抗CD40(克隆HM40-3),抗CD80(克隆16 -10A1)和抗CD86(克隆GL1))和适当体积的˚F低血细胞计数小号泰宁b uffer达到一最终染色体积的100 μ升。涡流。
孵育用于在冰上至少30分钟。避光。
通过加入2M洗细胞升流Ç ytometry小号泰宁b uffer到每个管中。
通过在250离心细胞重复洗涤步骤两次×克为在室温下8分钟。
除去上清,重悬在100沉淀μ升流Ç ytometry小号泰宁b uffer。
将细胞悬浮液转移到聚丙烯管中进行流式细胞术。
有关存储在分析之前样品,添加100 μ升IC ˚F ixation b uffer。
ANALY ž Ë的细胞根据荧光强度的上述标记和获取代表图像用于后续的分析/孔定量吨通货膨胀。


的沉默活性的评价抗-CD40-siRNA转澈在体内米乌斯模型
要求:G loves,手术刀,剪刀,注射器,毛巾,棉布,样品收集管,23 – 25 G针,小鼠,siRNA,PS缓冲液(参见R ecipes),异氟烷,异氟烷腔,便携式液氮容器,液氮,和采血管。


分离小鼠到治疗组和对照组。
研究在体内的全身给药的效果CHOL -siRNAs:使用6 - 8 -周大的雄性ICR小鼠(每组4只小鼠)。ADMINIST ER 50微克抗-CD40-siRNA转澈或S / S-控制的siRNA澈通过我。p 。注射(0.2 µm过滤的PBS中10 ml / kg )。在预定的时间点(天0,1,3,5,7 ,和9) ,从辖单剂量的5个微克LPS大肠杆菌通过我。p 。注射。给予LPS后4小时对动物实施安乐死。
对于5个月大的NZB / NZW F1狼疮肾炎研究,小号等了以下几组:
CYP:甲dminist ER 50mg / kg的我。p 。每10天(n = 9)。
CTLA4 :ADMINIST ER 50微克阿巴西普(ORENCIA ,Bristol Myers Squibb的)THR EE次每周(N = 9) 。
siCD40-1w :ADMINIST ER 50 μ克抗- CD40-siRNA转澈我。p 。每周一次(n = 9)。
siCD40-2w :ADMINIST ER 50 μ克抗- CD40-siRNA转澈我。p 。每周两次(n = 9)。
控制:ADMINIST ER 50 μ克S / S-控制的siRNA澈我。p 。每周两次(n = 15)。
对于与8周龄雌性ApoE -/-小鼠进行的硬化研究。牛逼不动产资产信托小鼠每周两次我。p 。用50 μ克抗- CD40-siRNA转澈,S / S-控制的siRNA澈或媒介物(每组5只小鼠)。安乐死的8周(基础组),10周,14周动物,或24周。
组织收集:尿液和静脉血。
将在个体代谢笼中的动物与水和在平时的饮食,并收集处理来临之前,之后每隔一个月24个小时尿液标本。
注意:如果小鼠在给定时间内没有产生尿液,则在第二天在更暖的房间中重复该步骤。


将尿液样品以500 × g离心10分钟。收集的尿液,并保留了进一步研究的沉积物; 存储的尿和沉积物在-20 ℃下。
每月两次确定小鼠的体重。
ø N A按月,使用短执行从尾静脉的非末端静脉采血(无需更换流体的)-term麻醉通过吸入异氟烷。
要记住的动物舒适性要点:


使动物舒适的温度保持在24 - 27 ℃的。
不擦的从尖端的基部作为尾部这将导致亮氨酸ķ ocytosis 。如果看不到静脉,则将尾巴浸入温水(40 °C )中。
插入一个23 - 25号针入使用毛细管或注射器针头的血管和采集血液。在困难情况下,切一个皮肤的最小表面,刺的与刺血针出血或针,并收集血液的毛细管或具有针头的注射器中静脉。
收集血液的次数不要超过三遍。每只小鼠的最大采血量应为0.2毫升血液。所估计的血液体积在成年动物为55 -为70ml / kg体重(巴拉苏罗等人,2010)。
完成采血后,停止了流血使用压力。
洗的频繁约束以避免信息素诱导的应力或交叉感染。
在研究结束时,通过心脏穿刺提取动脉血。
在异氟烷腔中对动物进行终末麻醉。
打开的胸部(胸廓),并且直接从心室获得的血液样本,同时注意不要崩溃的心脏。
组织收集:肝和肾
通过吸入异氟烷对所有小鼠实施安乐死。
解剖出了通过腹部肾脏和肝脏并通过灌注冰把它们洗干净-冷的1 ×经由左心室PBS。
切除肾脏。一个肾脏将用于组织学评估,另一个肾脏用于总RNA提取。
对于组织学分析,除去的上三分之一的肾脏,以确保两个皮层和近髓质肾小球存在并固定在5ml缓冲液PS的片在4 ℃下24-48小时。
对于免疫荧光分析,将三分之一的所述肾片(以确保两个皮质和髓质肾小球存在)进入组织模具和外衣在OCT中。将其绑在干冰上以冻结并储存在-80 °C下。
对于蛋白质和RNA提取,将3 × 2 mm 3的肾皮质和肝脏碎片放入0.5 ml塑料管中,并在液氮2中速冻。储存在-80 °C 。对于长期储存的组织进行RNA提取,放置了在RNA的5个体积组织稳定化试剂,并储存于-80 ℃下。
从肾脏组织中提取和纯化RNA。
要求:d isposable手套,升aboratory ˚F UME ħ OOD,无RNase的移液管和移液管尖端小号,无菌塑料器皿,冷TRIZOL TM ,转子-定子匀化器(ULTRA-TURRAX TM ),冰,圆底ED无RNase的管用于组织均匀化,无RNase水(用DEPC处理过的水,请参见[R eceip小号),桌面离心机,2毫升RNA酶-free Eppendorf管,ñ ANO d ROP ,的PureLink RNA mini试剂盒,ç hloroform,70%è THANOL(100 %乙醇与无RNAse的水混合),v涡旋。


1毫升添加冷的TRIzol 50 - 100毫克肾组织在一个圆底ED无RNase的管,搅匀在冰上。将探针的尖端浸在裂解液中,同时将探针的尖端紧贴在管壁上,以免产生泡沫。
孵育5分钟- [R OOM温度以允许的核蛋白复合物完全解离。
添加0.2米升氯仿并涡旋。
孵育2 -在室温下3分钟。
离心样品在12 15分钟,000 ×克,4 ℃下。
将混合物分离成一个较低的红酚-氯仿相,相间和无色上层水相。小心地将水相转移到一个2 -毫升Eppendorf管中(重要:避免传输任何相间或有机层的)。
加入一体积的70%乙醇并涡旋。
转印≤ 700 μ升样品到自旋Ç artridge并根据该净化总RNA试剂盒说明书(在此情况下的PureLink RNA迷你试剂盒)。
孔定量FY总RNA使用的Ñ ANO d ROP 。
第一链DNA合成(逆转录)。
要求:我CE,无RNase水,台式离心机,Eppendorf管,高容量cDNA逆转录试剂盒,热循环仪,70%ë THANOL,v ortex。


逆转录反应是根据所用试剂盒(在这种情况下为高容量cDNA反转录)的说明,使用500 ng RNA进行的。


Determin ê在CD40表达的肾脏和肝脏通过qPCR。
用CD40引物(Mn_00441891_m1; Taqman基因表达测定法,来自ABI / ThermoFisher Scientific,Waltham,MA,USA)建立反应。


分析抗- CD40-siRNA转澈-执导由5'RACE裂解产物。
要求:G爱,总RNA(来自C1节),热循环仪,cDNA合成试剂盒,5'RACE试剂盒。


合成第一链从5的cDNA μ克总RNA (肾)使用高容量cDNA RT试剂盒与基因特异性引物#1(GSP1):5' - GCCGACTGGGCAGGGATGACAGACG 。
将5'RACE试剂盒中的衔接子寡核苷酸连接到cDNA的5'末端。
特别是使用AMPLIFY裂解产物一个到适配器(5'RACE试剂盒)互补的引物和所述嵌套基因-特异性引物#2 (GSP2):GSP2:5'-AGCCAGGGATACAGGGCGTGTGC。使用以下PCR程序:1 m,94 °C × 60 s和94 °C × 30 s,55 °C × 30 s和72 °C × 60 s的35个循环,最后扩展为72 °C × 5分钟
检查是否存在310 bp扩增产物的存在,该产物对应于被抗CD40 siRNA特异性切割的CD40 mRNA。
使用20 μ升在1%溴化乙锭的PCR产物进行分析,溴化-染色的琼脂糖凝胶通过电泳与相应的DNA分子量标记物。


siRNA处理的动物肾脏病变的组织学评估。
在本节中,我们将准备肾样品为标准组织学分析(在石蜡中包埋),免疫荧光分析(OCT) ,以及蛋白质和RNA分析(存储在RNA stabiliz通货膨胀溶液)。


要求:一个在部分D2,手套,冰得到的肾脏-冷的1 × PBS,组织模具和盒,镊子,石蜡,石蜡分配器,冷板,PS缓冲液(见ř ecipes),15%小号ucrose,4%多聚甲醛(PFA),OCT,干冰,RNA stabiliz通货膨胀试剂,高-密度聚乙烯(HDPE),50%乙醇,70%乙醇,96%乙醇,无水乙醇,去离子水(中号IL升的iQ或类似的),切片机。


用于标准组织学分析。
除去肾的上三分之一,以确保两个皮层和近髓质肾小球存在并固定在5ml缓冲液PS的片在4 ℃下进行24 - 48小时。将薄页纸放入带标签的盒中(使用铅笔,因为溶剂会溶解墨水),并准备进行石蜡包埋。


浸盒在宽-嘴高-密度聚乙烯(HDPE)1大号罐填充有15%蔗糖24在石蜡块中嵌入前小时。
浸在50%乙醇2 - 3天。
浸在70%的乙醇对一个最小的3小时(但可维持高达至7天)。
浸入96%乙醇中过夜。
浸入无水乙醇中至少3小时。
用清除剂(洗涤二甲苯)为在室温下1个小时。
在60 °C下执行第一个石蜡整夜。
执行第二石蜡在60 ℃下为最小的3小时。
P花边在熔融石蜡少量模具(分配从一个石蜡容器)。用温暖的镊子,转移的组织进入模具切割面朝下,因为它是放置在盒中。
传送所述模具到冷板并轻轻按下的组织平面。石蜡将在薄薄的层中固化,从而固定组织位置。
当组织处于所需方向时,将标记的组织盒作为衬板添加到模具的顶部。用力按。
将热石蜡从石蜡分配器添加到模具中。可以肯定的是有足够的石蜡覆盖塑料盒的脸。如有必要,请在纸盒用石蜡一边冷却,一边保持模具全固体为止。
石蜡solidif IES在30分钟内。石蜡块然后可以被移除和部分编。如果蜡裂纹或组织不能很好对齐,再次融化他们,并开始了。组织块可以在室温下保存多年。
使用切片机(Leica RM 2155)切片组织。
打开水浴,检查温度是否为45 °C 。使用1升新鲜的去离子水,并从猪皮中加入1克果冻(G1890,Sigma-Aldrich,圣路易斯,美国密苏里州)。放置在切片机新鲜叶片(叶片可被用于以部分最多10块,但如果切片成为问题替换)。刀片应与5 °成d角。到被分段的块被面朝下放置为约15分钟冰块上(冷蜡允许较薄部分)。切后亭,使用镊子拾取部分的色带和它们漂浮在水浴中的水的表面上,以允许对所述样品的膨胀。
插入块与蜡块FAC切片荷兰国际集团的叶片,并对准垂直平面。拨盘设置为切口10 - μ米区段中以平面中的块; 一旦切割顺利,设置为2 - 3 μ中号厚的部分。通过将其切至所需的组织平面面向块,并丢弃石蜡带。
如果块状条带扎好,则再切成四部分,用镊子或细油漆刷将其捡起,然后将其漂浮在45 °C水浴中的水面上。将这些部分漂浮在干净的玻璃载玻片表面上。
如果色带不能很好地条带化,请将其放回冰块上冷却更长的时间以硬化蜡。
如果在放置标本支离破碎我n中的水浴中,那么它可能是太热了。
放置变暖块上的载玻片在一个60 ℃的烘箱中10分钟(因此蜡开始熔化)至键的组织到玻璃上。载玻片可在室温下保存过夜。
用于免疫荧光分析。
将肾脏的另一极放入组织霉菌中,涂上华侨城(OCT),然后将其放在干冰上冷冻。储存在-80 °C 。


P花边少量熔融OCT的(吨在问题冷冻培养基,徕卡价= 14020108926,徕卡生物系统公司,里士满,IL)模具。
使用镊子,转移的组织的模具,切割面朝下。
P我们的液氮2在以聚苯乙烯盒含有50 -毫升管架(不不在液氮淹没管架2 )。放置OCT模具上的管架和FR EE泽使用的蒸汽。
用箔纸包裹,并在液体N 2中速冻,直至在-80 °C下储存。
用于蛋白质和RNA提取。
代替3- × 2毫米3个肾皮质的成0.5 -毫升的塑料管中并咬合在液氮中冷冻2 。储存在-80 °C 。对于长期储存的组织进行RNA提取,放置在在RNA稳定试剂和存储的五个卷组织在-80 ℃下。


在IgG和C3存款的直接免疫荧光分析的肾脏。
要求:OCT包埋肾样品,手套,冰-冷的1 × PBS,低温恒温器,聚-L-赖氨酸-包被的载玻片(默克KGaA公司,CATA登录号:P0425) 。或FLEX IHC显微镜载玻片(安捷伦,参考K8020,圣克拉拉,CA,USA),4%PFA(见ř ecipes),丙酮,封闭溶液,缀合的初级抗体(FITC缀合的山羊抗小鼠IgG,FITC缀合的山羊抗小鼠C3),UltraCruz的Tm米ounting米edium ,和DRAQ5。


准备用于免疫荧光分析的肾脏玻片。
切片前,将包含冷冻肾脏的OCT复合模具(D3部分)在-20 °C下放置2 h。确保将肾脏的[切开]表面小心地靠在OCT模具的底部,以使组织切片方向正确。
使用低温恒温器切断为5μm厚的切片,并将它们聚-L-赖氨酸的表面上-包被的载玻片或FLEX IHC显微镜载玻片上。确保其没有褶皱或孔在组织部分,其可以变形的组织形态。作为IF阳性对照,使用人腮腺或神经节样本; 和作为阴性对照,省略了第一抗体从染色处理。对于同种型对照,使用相同同种型,物种的无关免疫球蛋白,和浓度作为初级抗体。
当从低温恒温器去除,保持在室温下20分钟,然后固定该滑动通过浸没入冰冷的纯丙酮20分钟。
洗的用100μlPBS载玻片3次为每次10分钟。
洗的载玻片两次用蒸馏水对每次5分钟。
将玻片在封闭溶液(20%正常山羊血清的1 × PBST + 0.2%的猪皮果冻中)中于4 °C孵育过夜。
洗的用100μlPBS载玻片3次为每次10分钟。
在含有1%正常血清的PBST-果冻缓冲液中,将这些切片与一抗(FITC偶联的山羊抗小鼠IgG以1:300和FITC偶联的山羊抗小鼠C3在1:50)孵育。孵育的载玻片为1个在室温下H以加湿室中以避免所述组织变干,其中W乌尔德导致非特异性结合和高背景染色。
洗的载玻片3次,在PBS中为每次5分钟。一抗可以保存用于后续实验。
安装在带安装介质一滴样品含有1微克/毫升DRAQ5。


在CD40表达的分析的与辣根过氧化物酶(HRP)的免疫染色肾脏。
要求:G爱,石蜡块,高压锅,计时器,1 × PBS,0.01 M柠檬酸盐缓冲液(pH 6),PBS中的1%Triton X-100、0.1%鱼胶,牛血清白蛋白(BSA),二甲苯,无水乙醇,初级抗体(最佳稀释度和温育时间应被确定为使用之前每一级抗体),生物素化的第二抗体(135 μ升正常血清+ 45 μ升生物素化的二级抗体来自ABC染色ķ它在10毫升PBS),加湿室,ABC(一和素-生物素复合物)过氧化物酶的标准染色试剂盒(制备使用前试剂30分钟),DPX封固介质,DAB底物。


对照:P rep单独用稀释剂(不含抗体)作阴性对照,用已知含有目的抗原的组织作阳性对照。


准备用于HRP分析的肾脏玻片。
Deparaffinize的从样品小号通过执行标准二甲苯-乙醇洗涤挠度D5。
洗的载玻片3次,在1%PBS对于每次5分钟。
阻止与甲醇洗涤(30%甲醇的PBS内源性过氧化物酶活+ 1%过氧化氢)为10分钟。
注意:将幻灯片放在平坦的表面上。不要让该幻灯片互相接触。不要让这些部分变干。


洗三次,在1%PBS对于每次5分钟。
促进抗原修复通过加热的高压锅的样品上5分钟(在塑料机架幻灯片)含有10mM柠檬酸盐缓冲液,pH 6,这将破坏福尔马林固定后蛋白质交联。
让其在室温下缓慢冷却30分钟。
在1%的Triton X-100的PBS(PBST)洗涤两次对于每次5分钟。
为了最大程度地减少交叉反应并减少由疏水相互作用引起的非特异性结合,请在4 °C下与PBS-Triton + 0.2%的果冻中的20%正常山羊(NGS)或马血清预孵育2 h。用轻快的动作从滑片上清除多余的液体,并仔细擦拭各个滑片。
;与第一抗体孵育(抗NF-抗CD40以1/100,抗-DC-SIGN在1/50 κB在1/1 ,000)在1%正常山羊血清。在每个玻片上加100 µl,覆盖组织切片;吨ILT每个载玻片从一边到另一边。在湿度箱中于4 °C孵育过夜。
在室温下保持30分钟。
在1 × PBST中洗涤3次,每次5分钟。
加入100 µl生物素化的二抗(以PBST-jelly中的Vectastain ABC试剂盒的浓度为1/200 + 1%NGS )。在室温下在加湿室中孵育至少45分钟。在该步骤期间,建议制备底物混合物(试剂A =一个维丁+试剂B = b iotinylated HRP)。
在PBST中清洗3次,每次5分钟。充分洗涤以除去痕量叠氮化钠,因为这将在显影时抑制过氧化物酶的活性。
加入底物混合物,比例为1/100,孵育45分钟。
在PBST中清洗3次,每次5分钟。
用PBS洗涤3次,每次5分钟。
孵育所述组织部分小号与在黑暗中3分钟或直至所需的50微升矢量DAB底颜色时监测反应观察下的显微镜。通过用洗瓶中的蒸馏水轻轻冲洗,终止反应,然后在阴性对照中出现背景染色。在相应的收件人中释放DAB。所有的塑料制品而来的对与DAB的接触必须用稀释的漂白处理。
用自来水冲洗5分钟。
用蒸馏水洗涤。
用苏木精复染:
加入苏木精(1份苏木精溶液+ 2份蒸馏水)。
用自来水清洗5分钟。
用蒸馏水洗涤。
                                                                                                                                                                   用70%乙醇洗涤5分钟。
执行三次洗涤用96%乙醇对每次5分钟。
用无水乙醇进行洗涤三次为每次5分钟。
执行洗涤三次用二甲苯对每次5分钟。
用DPX挂载。
显微镜检查:
切片应由两名不知情的病理学家独立检查,每个肾脏切片至少应有10个视野在高放大倍数下进行研究。


确定肾脏损害的程度。驴小号狼疮性肾炎的典型肾小球活性病变:肾小球系膜扩张,毛细血管内增殖,肾小球存款,外毛细管增殖,和间质浸润; 以及肾小管间质性慢性病变:使用Carl Zeiss AG(德国Oberkochen ,德国)的Zeiss SteREOLumar V12显微镜观察肾小管萎缩和间质纤维化。级的病变半定量使用评分系统0〜3(0 =没有变化,1 =轻微,2 =中度,3 =重度变化)。
孔定量泰特的数量使用半研究了每种标记物的阳性细胞-表达的定量评价从0到4(0 =没有染色,1 =在该样品的<25%染色,2 =在25染色- 50%, 3 =染色在50 - 75% ,和4 =在75染色-在肾(肾小球,容器的不同隔室100%) ,和间质)。NF κB位于细胞核内,当P65免疫染色阳性。


在米粥样硬化病变的评价乌斯主动脉。
要求:ApoE基因- / -小鼠中,异氟烷室,清扫工具(剪刀:直钝-ended剪刀,直锋利细剪刀,和微-解剖弹簧剪刀;组织钳:直锯齿-尖镊子,直的或弯曲锯齿-尖端细钳子,和直细-尖镊子),注射器,23 G和27个针头,管(一个EDTA涂覆的试管中,血清管,以及两个锂肝素包被的管),冰-冷的PBS,去离子水,RNAzap , 70%的乙醇,冰,液氮2 ,显微镜,软木床,钉扎床,针,手术刀,ö IL ř编ø ,minutien引脚0.1 -毫米,培养皿,6 ×放大倍数的显微镜。


检查整个主动脉长度中ATH病变的扩展。
用异氟烷对小鼠进行安乐死。
用70%的乙醇喷洒每只小鼠,以免污染样品。
切除整个主动脉(根据Centa等人的方法修改,2019年)并准备用于显微镜分析的样品:
用剪刀从颈状切口切开至耻骨。
通过穿过胸壁的心脏穿刺使小鼠放血(使用23 G针)。此过程通常会产生500 μ升从血液一个20周龄的小鼠。收集4个管(一个EDTA涂覆的试管中,血清管,以及两个锂肝素包被的管)。将其保持在室温下。
打开腹腔。用剪刀在中线和两侧横向切开腹膜腹膜。 
打开隔膜和胸腔(尽可能从侧面切开肋骨保持架)。
在右耳中切开切口,以排出灌注液。通过心脏的心尖插入一个27号针的颅方向。保持固定在左心室,同时缓慢用10ml冰冷的PBS灌注针在一个最小的2分钟。
解剖的肝脏,脾脏和肾脏。
切开心脏右侧的气管和食道,而不会损坏主动脉弓。切割膜片和结构附连到内脏的腹膜后。离开的心脏,主动脉,和肾脏原位。折叠起来肺和内脏尾端和盖用餐巾纸开始的腹膜后显微切割的腹部大动脉(下进行显微解剖一个在6显微镜×放大率)。
解剖主动脉分叉(解除了与钳在张力下周围组织和用剪刀剪)。颅骨解剖腹部主动脉。切的从主动脉腹部分支和通过隔膜主动脉裂孔近侧释放主动脉。
去除覆盖胸主动脉的脂肪组织。仔细解剖胸腺背侧,以释放带有分支的主动脉弓。继续在胸腔内尽可能向远侧解剖颈动脉。颈部解剖应包括颈动脉分叉。
在切断主动脉之前,先在去离子水,RNase净化溶液,70%乙醇和PBS中依次冲洗,以清洁仪器。
用镊子将心脏提起。切开靠近心脏的主动脉,然后将整个心脏放入装有PBS的试管中。的心脏可以被存储在冰上几个小时之前continu ING与处理和低温安装的主动脉根部。
在含有1 ml 4%甲醛的试管中于4 °C过夜,将主动脉弓和花边切成两半。
解剖剩余降主动脉,p花边在管,并在液氮中冷冻单元2用于RNA分析。
将心脏放在软木床上,使腹侧朝上。用针头穿过顶端将心脏固定在软木塞上。用解剖钳固定心脏的底部。切去两个心房之间的心脏的心尖2/3用手术刀(倾斜20 °在矢状面和尾部20 °颅在横向平面)。
将主动脉根嵌入OCT化合物中(请参阅F节)。将标本保存在80 °C的拉链锁袋中,直至进行冷冻切片。
“ ê的N面”分析了主动脉弓和braquiocephalic动脉。
清洗从剩余的外膜周的脂肪组织的主动脉下一个显微镜(因为油红O污渍最疏水的和中性脂质如甘油三酯,甘油二酯,和胆甾醇酯,这是至关重要的在这一点上,以除去所有这样的组织)。使用剪刀和镊子,不要操纵或损坏主动脉。始终在需要时通过使用额外的PBS来保持主动脉湿润。
放置在清洁和固定主动脉一个1.5 - ml管(每管一个主动脉)。
向每个试管中加入1 ml 78%的甲醇,并在倾斜的滚筒上轻轻移动5分钟。更换甲醇溶液,然后重复此步骤两次。
丢弃甲醇,并加入1毫升新鲜的Oil RedO 。
孵育倾斜辊上的管50 - 60分钟。
转移主动脉干净的管中并用1ml 78%的甲醇洗两次,每次5分钟的翻转机罗尔ř 。
丢弃甲醇,并用1 ml PBS重新填充试管。
注意:如有必要,在此步骤中,主动脉可以保存在4 °C下。


准备一个钉扎床:将25片× 25毫米石蜡的蜡膜,包裹着黑色电人绝缘胶带,在软木床,以便为主动脉一个黑暗的背景。在固定床的背面放置一个标签,并使用一支铅铅笔写下鼠标的识别号(正常的笔墨水会在染色过程中消失)。
将固定的主动脉弓转移到固定床上,并在上面放一滴PBS。
除去外来脂肪的任何少量残留的染色剂(通过显微镜检查仔细观察)。
纵向切开主动脉以暴露内膜表面。引进一对微的尖端-解剖弹簧剪刀到动脉腔和从上升拱到左锁骨下动脉切开主动脉弓的外曲率。开始削减外上升圆弧的曲率在所述远侧方向和继续切开分支,包括头臂动脉。Ç ontinue到沿胸主动脉的长度切断。
切开较小的曲率,然后折叠打开主动脉以显示内膜表面。
使用Minutien昆虫图钉的钝端将打开的拱形图钉在固定床上。到位后,将插销轻轻弯曲,使其远离样品。将主动脉平放在床上,而不拉伸标本。将固定的弓形朝下存放在4 °C的装有PBS的培养皿中。
采集我法师用Zeiss steREOLumar连接到RGB相机V12显微镜(PROGRES CF扫描)。将标尺放在主动脉旁边以校准图像。
孔定量泰特atherosclero的延伸抽动斑块。
使用图像分析软件(ImageJ的),以确定所述损伤面积和总内膜表面。大号esion孔定量吨通货膨胀应执行以盲方式,并且它是可取的第二研究者确认š结果。


计算拱的总面积:在ImageJ中,选择“多边形选择”工具,然后通过重复单击来包围拱的总面积。在选择“度量” ANALY ž ê菜单显示在结果窗口中的总足弓部位。
计算无病变区域:在ImageJ中,选择“徒手选择”工具,并在按住Alt键的同时将所有斑块(橙红色染色)包围在拱形区域中。单击“度量” ANALY ž ê菜单显示了在结果窗口中自由病变区域。
计算的通过减去无病变从总足弓区域和分割结果相对病变区域由总足弓区域。
主动脉根的冷冻切片。
在-20设置在低温恒温器温度℃,并且截面厚度〜10 μ米。将包含主动脉根的OCT b锁安装在标本架上,并使心室组织朝外。在开始切割时,微调-调整截面表面的对齐方式,使其平行于样品架。
删除了周围多余的华侨城,使其更容易收集的部分无褶皱。主动脉根应垂直于刀片放置。
收集普通显微镜载玻片上的初始对照切片并丢弃。第一部分应仅包含心肌组织。进度由200切片μ米。收集切片并在光学显微镜下检查进度。
当越来越接近左心室流出道,检查每100 μ米的显微镜下。当观察到血管壁的初始指示,放慢步伐〜50 μ米。当第一个主动脉瓣出现时,这将是收集部位的零点。可能很难准确地看到瓣膜何时出现,但是准确的定位对于在相同区域进行病变比较至关重要。
将样品向零位阀倾斜,以使剖面与其他两个阀对齐。这是获得真正的跨至关重要-主动脉的部分。使主动脉根的图,表示阀,因为它们出现,并且计数每10 - μ是m部分被切断从点零向前。当出现第二个主动脉瓣膜时,再次从瓣膜上稍微倾斜样品以使样品与第三个瓣膜对齐。从第一主动脉瓣小叶出现在主动脉瓣叶的所有3出现在距离一起不超过80 μ米。前5 - μ微米厚部分与3个主动脉瓣叶被安装到载玻片上。相邻的8个部分(包括第一个部分)被一个个地收集到8个载玻片上,并按从1到8的顺序进行标记。第9个部分被收集到第一个载玻片上,第10个部分被收集到第二个载玻片上,依此类推类推,直到每个的8个载玻片š保持8个部分。在8个收集在每个载玻片上的冷冻切片覆盖的400的距离μ米。
将收集的切片在4%甲醛中固定用于油红O染色10分钟,并用冰-冷的纯丙酮固定10分钟以进行免疫组织化学或免疫荧光分析。在室温下干燥30分钟。存储的部分在-20 ℃下。
捕获的直接图像使用一个Ñ RGB相机(PROGRES CF扫描)附接至光显微镜(Nikon E800)。保存高分辨率图像,最好以标记图像文件(TIFF)格式保存。
使用ImageJ进行形态学图像分析。
病变孔定量吨通货膨胀应执行以盲方式,并且最好是使第二研究者证实了获得的结果。


使用面积孔定量牛逼通货膨胀功能在图像分析软件通过环绕主动脉血管的外弹力层定义总的血管面积。在ImageJ中,选择“多边形选择”工具,然后通过重复单击来包围区域。该ñ在选择“测量” ANALY ž ê菜单。总血管面积显示在结果窗口中。
继续孔定量泰特的ATH病变在容器的内膜层,由内弹性层和管腔边界限定。通常,将主动脉瓣上的病变排除在测量范围之外。在ImageJ中,选择“徒手选择”工具,并在按下Alt键的同时将所有斑块包围起来。在选择“度量” ANALY ž ê菜单显示在结果窗口中的无病变血管面积。
通过减去总血管区中的自由病变区域和分割结果计算相对病变区域由总血管面积。根据放大率校正在图像分析软件的结果,以便用得到的在绝对病变区域微米2 。


数据分析


              胆固醇缀合的,特异性的抗CD40的siRNA显示出有效的转染效率d endritic Ç厄尔(DCS),而不降低细胞活力(评价通过碘化丙啶)。这里,我们证明CD40表达的抑制和亚细胞轨迹莉莎和灰抗- CD40-siRNA转澈在DC的细胞质在45分钟后转染(Ripoll会面等人,2013 )。5'RACE证实了CD40 mRNA的siRNA切割的特异性。


  (在沉默实验Ripoll会面。等人,2013 ),(由证实我们首先成熟的DC用LPS的增加CD40,CD80 ,和CD86细胞表面表达如通过流式细胞术测量的),并转染它们与抗- CD40-siRNA转澈,这引起了35%在CD40的表达降低为与加扰控制,以及在TNF释放的显著相比减少 α,MCP1 ,与IL6。此外,增加LPS CD40 mRNA表达几乎20倍(在所述肾)和35倍(在所述肝脏)与比较在上午对照水平乌斯模型。CD40 mRNA表达回到基线后24小时的肾脏和后48在肝脏小时。单个抗- CD40-siRNA转澈给药65%和60%降低肾和肝CD40 mRNA表达,分别,3天为与对照值相比,具有效果持续长达5天。


  (在狼疮性肾炎模型Ripoll会面。等人,2013 ),Cy5.5标记的抗- CD40-siRNA转澈被定位在从肾小管细胞的肾脏(图1)。在这种疾病模型,我们研究的抑制作用抗- CD40-siRNA转澈对动物的生存,肾功能,炎症介质,抗DNA antibod Ÿ水平,以及肾脏病变。对于这些实验,将小鼠分布在给五组中的一个进行不同的处理:


CYP组(n = 9) :50mg / kg的我.P 。每10天CYP
CTLA4组(n = 9) :50微克IP CTLA4(ORENCIA,阿巴西普,Bristol Myers Squibb的,Uxbridge的,UK)每周三次
抗- CD40-siRNA转澈-1W组(n = 9) :50微克IP 。抗- CD40-siRNA转澈每周一次
抗- CD40-siRNA转澈-2W组(n = 8) :50微克IP 。抗- CD40-siRNA转澈两次一个星期
对照组(n = 15) :50微克腹膜内。SS -对照的siRNA澈两次一个星期




图1.在狼疮性肾炎模型,Cy5.5标记的抗- CD40-siRNA转澈被定位在从肾小管细胞的肾。A.孔定量牛逼的肾脏内的通货膨胀哲-siRNA给予我。v 。/我。p 。在狼疮小鼠中。B.代表肾显微照片(× 400) :1)b ASAL自发荧光; 2)我。v 。行政; 和3)我。p 。行政。数据表示为四个独立实验的平均值± SEM。* P <0.05 vs.基础。doi:10.1371 / journal.pone.0065068.g005。


动物存活(通过Kaplan - Meier法)为100%的CYP,CTLA4 ,和CD40-2w基; 随访结束时,CD40-siRNA-1w组为88%,未治疗组为73%。与siCD40-2w处理的小鼠显示蛋白尿或蛋白尿没有增加并显示一个剂量依赖性降低在总IgG,抗dsDNA抗体,以及所有的IgG级分。该升的IgM,IgA的的evels ,和IgE的,以及促炎细胞因子例如IL2,TNF,IFN γ ,MCP1 ,与IL6 ,也减少了在该抗- CD40-siRNA转澈-2W基。使用0到3的评分系统对组织学病变进行半定量评分(对照组= 8 ± 1.5; CYP = 4.3 ± 1.1 ; CTLA4 = 1.6 ± 0.7; 抗- CD40-siRNA转澈-1W = 3.8 ± 1.5; 抗- CD40-siRNA转澈-2W = 1.6 ± 0.6) ,以及IgG和C3肾小球沉积物在所有治疗组(图减少小号2和3)。抗- CD40-siRNA转澈-2W组显示出一个不存在的额外的毛细血管增生,间质浸润,肾小管萎缩,和间质纤维化。除siCD40-1w外,所有治疗组小管间隙中的CD3 +浸润细胞均显着减少。此外,肾脏表达水平的CD40,C3(局部补体合成的体现)的,以及促炎细胞因子瓦特ERE减少在抗- CD40-siRNA转澈-2W小鼠(图4)。另外,显著减少在CD40蛋白表达在间质,肾小球中观察到,和肾脏血管隔室和在所述circulati上中抗CD40-siRNA转澈-2W基。


  最后,我们使用CD40沉默策略来降低atherosclero的进展抽动(ATH)病变的ApoE - / -小鼠(Hueso等人,2016 )。我们分布在小鼠中,以8个治疗组:


基础B / 8w(n = 5)
ss- control siRNA Chol / 10 w(n = 5)
ss- control siRNA Chol / 24w(n = 5)
加扰的寡核苷酸对照SC / 14w(n = 5)
一个nti - CD40-siRNA Chol / 10w(n = 5)
一个nti - CD40-siRNA Chol / 14w(n = 5)
一个nti - CD40-siRNA Chol / 24w(n = 10)
车辆(n = 5)


与油红染色整个主动脉的“前位”分析Ø证实,ATH斑块面积的数量和延长反下降- CD40-siRNA转澈/24瓦特与比较控制值。少F4 /是在血管壁检测从抗80浸润巨噬细胞- CD40-siRNA转澈/24瓦特动物,这表明对于CD40在巨噬细胞的斑块的募集的作用。最后,NF-少κ乙+抗内膜检测细胞- CD40-siRNA转澈/24瓦特小鼠,这表明CD40沉默的保护作用可以通过NF-介导κ乙信令。由于策略旨在到全身烯丙基silenc ê CD40 ,在CD3的脾种群的减少+ CD40 + (T淋巴细胞)和CD11b + CD40 + (单核细胞)的细胞中观察到在抗- CD40-siRNA转澈/24瓦特动物,这表明在粥样硬化病变的减少中号AY与抗炎机制在血管壁相关联经由全身效应。




图2. Histopat ħ ological病变的由CD40的siRNA沉默狼疮性肾炎模型。A.共刺激封锁减少ð狼疮性肾炎病变。乙。代表性的显微照片(× 200)的各组肾脏的组织学。数据是快捷ED作为该平均值± SEM。* P < 0.05,与。未经处理,P < 0.01vs 。未经处理。doi :10.1371 / journal.pone






图3.免疫组织化学分析的肾IgG和C3中的由CD40的siRNA沉默狼疮性肾炎模型。肾IgG和C3的A.存款孔定量达ED通过共聚焦显微镜(MFI)。所有治疗均减少了肾小球沉积。B.代表p C3沉积物(的hotomicrographs × 630)为每个组。数据表示为在平均值± SEM。* P < 0.05,与。未经处理,P < 0.01vs 。未经处理。doi:10.1371 / journal.pone.0065068.g006 。




图4.全身循环炎性细胞因子和局部CD40免疫染色。A.狼疮性肾炎促进了CD40的表达中的血清; 这种免疫调节蛋白在所有疗法中都减少了。狼疮肾炎也引起其他炎症细胞因子的增加; 具有抗治疗- CD40的siRNA降低了其中的一些。B.免疫定位和孔定量吨在不同肾隔间的CD40蛋白质的通货膨胀; Chol-siRNA降低CD40表达,尤其是在间质细胞和血管中。数据表示为在平均值± SEM。一个P < 0.05,与。未经处理 B对。CTLA4 ; ÇVS 。CYP ; 和d比。siCD40-2w。doi:10.1371 / journal.pone.0065068.g007。


菜谱


退火b uffer 10 ×
1的1M Tris-HCl(pH 7.5)中5米升


5M的NaCl 2米升


DEPC处理过的水43米升


经DEPC处理的水
二pirocarbonate (DEPC)0.1%1米升


蒸馏水1 ,000米升


拌匀,并留在室温下1个小时


高压釜


使用前让其冷却至室温


1 M Tris-HCl,pH 7.5
Tris 121.14克


蒸馏水800米升


调整的pH值至7.5用浓HCl的适当体积


带来的终体积为1 l在去离子水


12%丙烯酰胺凝胶
对于15米升,足以让一个13厘米× 15厘米× 0.75毫米厚凝胶:


10 × TBE 1.5百万升


40%的丙烯酰胺(丙烯酰胺:双丙烯酰胺= 29:1)4.5米升


蒸馏水和去离子水9 m l


搅拌混合,将n添加:


10%过硫酸铵150 μ升


TEMED 15 μ升


简单地混合加入最后2种成分后,倒了立即凝胶


非-变性凝胶缓冲液
蔗糖50%


溴酚乙LUE 0.25%


二甲苯氰0.25%


PBS中的4%PFA溶液
注意:甲醛是有毒的


要求:手套,防护眼镜,˚F UME罩


地方PBS 1 × 800米升在搅拌盘上的玻璃烧杯中在通风橱
在搅拌的同时加热到60 °C (注意不要沸腾)
加40克低聚甲醛粉
通过从移液管中逐滴加入1 N NaOH缓慢提高pH值,直到溶液澄清为止
冷却溶液并过滤
用1 × PBS将溶液的体积调节至1 L
重新检查pH值,并用少量稀盐酸调节至6.9
溶液可以等分并冷冻或在2.8 °C下保存长达一个月
0.1 M柠檬酸盐储备缓冲液,pH 6
蒸馏水800米升


柠檬酸(MW = 192.1 g / mol)11.341 g


柠檬酸钠(MW = 294.1 g / mol)12.044 g


调节的溶液,以在利用HCl或NaOH最终所需的pH


加蒸馏水至1 ,000毫升


在室温下储存(保质期长达3个月)


0.01 M柠檬酸盐缓冲液
0.1 M柠檬酸盐缓冲液100 ml


蒸馏水至900毫升


完整Ç ulture米edium
RPMI 1640培养基500毫升


100U / ml的p enicillin


100 μ微克/毫升小号treptomycin


2 M L-谷氨酰胺


10%的热量-灭活并过滤的FBS


20 ng / ml GM-CSF


0.3%的油红Ø小号滴答小号olution
油红O 0.3克


异丙醇99%100毫升


将溶液在56 °C溶解1小时


用Whatman No.1滤纸过滤溶液并保持在4 °C


该解决方案稳定1年


油红O工作溶液
在通风橱中准备


股票小号olution 60毫升


蒸水40毫升


用Whatman 1号滤纸过滤溶液


溶液稳定时间不超过2小时,使用前必须准备15分钟


磷酸盐缓冲小号艾琳(PBS),pH 7.5中
0.1M的p hosphate


0.15 M氯化钠


PBS-Triton(PBST)
海卫一X 2毫升


PBS 1 ,000毫升


PBST /果冻


猪皮果冻0.1克


PBST 50毫升


PS缓冲
福尔马林10%200毫升


水,质量中号IL升的iQ或类似200毫升


热60 - 70 ℃下


加入1粒NaOH


在水中冷却至24 °C


加入37.5克D +蔗糖


加入2mL的0.5M EDTA和2.5ml BHT(b utylated hidroxytoluene )


用1 M NaOH调节pH到7.4


添加的MilliQ水至500ml和FILT ER使用滤纸与介质滤过率粒子保留的10 - 20微米


二抗(1:200稀释)
抗体0.5 μ升


血清Ñ ormal克燕麦(或马)20%1 μ升


PBST /果冻98.5 μ升


血清正常山羊或马20%
从正常山羊或马血清20 μ升


PBST / 80果冻μ升


致谢


这项研究是部分由资助研究所德Salud的卡洛斯三世(通过共同出资的通过项目欧洲区域发展基金。ERDF,一个方式来构建欧洲)PI11 / 00556,PI14 / 00762 ,和PI18 / 01108和REDinREN (12 / 0021)。我们感谢REDinREN和CERCA程序/ Generalitat德加泰罗尼亚机构支持。


利益争夺


没有利益上的冲突。


伦理


该实验按照动物实验与欧盟法律进行,分别由CEEA批准:动物实验伦理小号委员会,制度伦理委员会UB动物研究。


参考


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引用:Hueso, M., Mallen, A., Ripoll, E., de Ramon, L., Bolaños, N., Valera, C., Guiteras, J., Checa, J., Navarro, E., Grinyo, J. M., Cruzado, J. M., Aran, J. M. and Torras, J. (2021). In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining. Bio-protocol 11(10): e4032. DOI: 10.21769/BioProtoc.4032.
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