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Jan 2021
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In situ Hybridization of miRNAs in Human Embryonic Kidney and Human Pluripotent Stem Cell-derived Kidney Organoids
在人胚肾和人多能干细胞衍生的肾类器官中微小RNA的原位杂交   

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Abstract

MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by high-throughput methods such as next generation sequencing. However, this provides little information as to the distribution of the miRNAs in the developing kidney, which is crucial in deciphering their role, especially as there are multiple kidney cell types, each with its own specific transcriptome. Thus, we present a protocol for obtaining spatial information for miRNA expression during kidney development by in situ hybridization (ISH) of anti-miRNA, digoxigenin-labelled (DIG), Locked Nucleic Acid (LNA®) probes on (i) native human embryonic tissue and (ii) human pluripotent stem cell (hPSC)-derived 3D kidney organoids that model kidney development. We found that the method reveals the precise localization of miRNA in specific anatomical structures and/or cell types and confirms their absence from others, thus informing as to their specific role during development.

Keywords: Kidney development (肾脏发育), Organoids (类器官), hESC (人类胚胎干细胞), hPSC (人胰腺星状细胞), MicroRNA (微小rna), In situ hybridization (原位杂交), LNA probe (LNA探针)

Background

MicroRNAs (miRNAs) are small (20-25 nucleotides) RNAs that regulate gene expression by binding predominantly to the 3’ UTRs of their target genes’ mRNAs and inhibiting their translation and/or causing their degradation (Bartel, 2018). An increasing number of studies now show that miRNAs play a crucial role in fine-tuning molecular pathways in the kidney, both in normal development and in disease (Jones et al., 2018; Trionfini et al., 2015; Zhao et al., 2019). The majority of these studies have been conducted on animal models, which do not always faithfully recapitulate the developmental events or disease phenotypes in humans. To overcome these limitations, we recently reported using human pluripotent stem cell (hPSC)-derived kidney organoids (Bantounas et al., 2018; Takasato et al., 2015) as a model to study the role of the miR-199a/214 cluster during human kidney development (Bantounas et al., 2021). In that study, we used in situ hybridization of miRNAs with digoxigenin-labelled locked-nucleic acid (LNA®) probes to detect the precise localization of the miRNAs of this cluster in paraffin sections of both native kidney and organoids. LNA® probes have a chemically modified backbone to afford a higher melting temperature (Tm) per nucleotide than conventional DNA or RNA probes (Singh et al., 1998). They are, thus, ideally suited to the detection of smaller RNAs for which conventional probes would have too low a Tm to bind efficiently. After binding to the tissue, the probes are detected by application of an alkaline phosphatase (AP)-linked, anti-digoxigenin antibody, followed by the addition of an AP substrate, which is converted to a coloured product, visible under a light microscope. Using this method, we discovered that miR-199a-3p and miR-214-3p were both present in the kidney stroma and in developing glomeruli but were largely absent from mature glomeruli. One of the miRNAs, miR-214-3p, also exhibited strong tubular expression. In addition, we observed differences in the extent and distribution of expression between embryonic/fetal kidney and organoids (particularly in the case of miR-199a-3p), possibly indicating that the two represented different developmental stages (Bantounas et al., 2021). This example demonstrates the main advantage of this method, which is the acquisition of spatial information regarding the expression of the miRNAs of interest within a given tissue. This information would be lost if one were to rely exclusively on transcriptional detection methods [e.g., qPCR or Next Generation miRNA Sequencing (miRseq)]. The hybridization steps of the protocol presented here (see below) were adapted from Jørgensen et al. (2010) and were optimized for kidney/kidney organoids and the particular miRNAs we studied (Bantounas et al., 2021). However, the method can in principle be used with formalin-fixed, paraffin-embedded (FFPE) sections of any tissue and for any miRNA or other small RNA of similar size.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes (Starlab, catalog number: S1615-5550)

  2. Plastic pipette tips (preferably filtered) (Starlab, catalog numbers: S1122-1830 [1,000 μl]; S1120-8810 [200 μl]; S1123-1810 [20 μl]; S1121-3810 [10 μl]; or equivalent)

  3. Vacuum filter units, pore size 0.2 μm (Thermo, catalog number: 568-0020)

  4. 6-well plates (Costar, catalog number: 3516, or equivalent)

  5. 3 ml plastic Pasteur pipettes (Starlab, catalog number: E1414-0311, or equivalent)

  6. Tissue processing/embedding cassettes (Simport, catalog number: M490-4) (Figure 1, Left)

  7. Stainless steel or plastic tissue processing capsules (Figure 1, Right)

    Note: The capsules depicted in Figure 1 were the ones used by the authors but have now been discontinued. Therefore, alternative options are proposed here: Simport, catalog number: M470; or Fisher, catalog number: 15-182-219; or equivalent.



    Figure 1. Tissue processing cassette and capsule


  8. Metal molds for paraffin embedding of tissue (Leica, catalog number: 3803081E)

  9. RNaseZap® wipes for disinfection of working surfaces (Invitrogen/Ambion, catalog number: AM9788)

  10. Superfrost® Plus slides (ThermoFisher Scientific, catalog number: J1800AMNZ)

  11. Human embryonic kidney tissue was provided by the MRC and Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org/)

  12. hPSC-derived kidney organoids can be produced according to published protocols (Takasato et al., 2015; Bantounas et al., 2018 and 2021). In our study (Bantounas et al., 2021), we differentiated the MAN13 human embryonic stem cell (hESC) line (Ye et al., 2017), but any hESC or induced pluripotent stem cell (iPSC) line can be used in principle

  13. Paraffin wax for embedding tissue, melting point 61°C (Pfm Medical, catalog number: 9000)

  14. Nuclease-free water (not DEPC-treated) (ThermoFisher Scientific, catalog number: AM9930)

  15. Phosphate buffered saline (PBS), without Ca2+ and Mg2+ (Sigma, catalog number: D8537-500 ml)

  16. 5’-Digoxigenin-labelled miRCURY® LNA® miRNA detection probes (1 nmol) (see Note 1) (QIAGEN, catalog number: 339111; see below for individual probe codes). In this example, we used probes targeting:

    1. miR-199a-3p (QIAGEN, catalog number: YD00615410)

    2. miR-214-3p (QIAGEN, catalog number: YD00611471)

    3. scrambled probe (negative control; QIAGEN, catalog number: YD00699004)

    4. U6 snRNA (positive/optimization control; QIAGEN, catalog number: YD00699002)

  17. miRCURY® LNA® miRNA ISH Buffer Set (FFPE) (QIAGEN, catalog number: 339450), which includes:

    1. 2× Formamide-free miRNA ISH buffer

    2. Proteinase K solution

  18. UltraPureTM 20× SSC Buffer (Invitrogen, catalog number: 15557-044)

  19. Tween-20 (Sigma, catalog number: P1379-250ML)

  20. Sheep anti-DIG-AP (alkaline phosphatase linked) antibody (Roche/Sigma-Aldrich, catalog number: 11093274910)

  21. Sheep serum (Sigma-Aldrich, catalog number: S3772-10ML)

  22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1470-100G)

  23. Nitro blue tetrazolium/5-bromo-4-chloro-indolyl-phosphate (NBT/BCIP) ready-to-use tablets (Roche, catalog number: 1 1 697 471 001)

  24. Levamisol (Fluka, catalog number: 31742), diluted in water to make a 100 mM stock solution

  25. Nuclear Fast Red solution (Merck, catalog number: N3020)

  26. Industrial methylated spirit (IMS) (various suppliers)

  27. Xylene (various suppliers)

  28. 100% Ethanol (various suppliers)

  29. KCl (various suppliers)

  30. KP Marker Plus (Histolab, catalog number: 98307-R) or similar hydrophobic marking pen (various suppliers)

  31. 4% Paraformaldehyde (PFA) Solution (see Recipes)

  32. 1 M Tris Buffer (see Recipes)

  33. 0.5 M EDTA Solution (see Recipes)

  34. 5 M NaCl Solution (see Recipes)

  35. 1× Proteinase K Buffer (see Recipes)

  36. SSC Buffer dilutions (see Recipes)

  37. PBS-T 0.1% (see Recipes)

  38. Blocking solution (see Recipes)

  39. Antibody dilution buffer (see Recipes)

  40. KTBT Buffer (see Recipes)

Equipment

  1. Tissue Processor (Leica, model: ASP300S; or equivalent)

  2. Heated Paraffin Embedding Station (Leica HistoCore Arcadia H or equivalent)

  3. Cold Plate (Leica, HistoCore Arcadia C or equivalent)

  4. Microtome (Leica, model: RM225; or equivalent)

  5. Hybridization oven or other variable temperature incubator (e.g., Hybaid, Midi Dual 14 or equivalent)

  6. Microscope slide rack (Simport, catalog number: M905-12DGY)

  7. Jars for immersing the slide rack into solvents/buffers (Simport, catalog number: M900-12G)

  8. Heating block (up to 90°C) (Eppendorf ThermoMixer F1.5 or equivalent)

  9. Microcentrifuge (Labnet, Prism or equivalent)

  10. Gilson® or equivalent pipettes (various suppliers)

  11. Staining tray for holding the slides during hybridization and staining (see Figure 2).

    Note: The tray depicted in Figure 2 was the one used by the authors but has now been discontinued. An alternative is proposed here: Fisher, catalog number: 22-045-035.

  12. Waterbath (Grant SUB6, catalog number: P266; or equivalent)

  13. Autoclave (various suppliers)

  14. Optionally, for imaging: 3D-Histech Panoramic-250 microscope slide-scanner, with a 40×/0.95 Plan Apochromat objective (Zeiss)

Software

  1. Optional: CaseViewer (3DHISTECH Ltd.; www.3dhistech.com), to capture images following scanning with a 3D-Histech slide-scanner (see point 14, in Equipment)

  2. Fiji/ImageJ (http://imagej.net/Fiji/Downloads) (for image processing following capture)

Procedure

  1. Tissue Fixation

    The procedure described in this section is for preparing organoid tissue and is an adaptation of the one described in Lopes et al. (2019).

    If beginning with samples already embedded in paraffin: Skip directly to section B, step 10.

    The procedure below assumes organoids cultured in transwell inserts in 6-well plates (Bantounas et al., 2018). If a different size well and/or insert is used, change volumes proportionally in Steps A1-A7.

    1. Prepare “wash” and “fixation” 6-well plates, allowing one well per transwell insert of organoids:

      1. Wash plate(s): Add 1.2 ml PBS into each well.

      2. Fixation plate(s): Add 1.2 ml of 4% PFA into each well.

    2. Transfer the transwell inserts from the culture plate to the wash plate prepared above.

    3. Carefully add 1 ml of PBS into each transwell and swirl the plate gently to wash the organoids.

    4. Using a Gilson or a 3 ml Pasteur pipette, remove the PBS from inside the transwells.

    5. Transfer the transwells from the “wash” to the “fixation” plate (i.e., on top of the PFA that is already in each of the wells).

    6. Add 1 ml 4% PFA into each transwell making sure that the organoids are fully covered.

      Note: You may add more PFA, if necessary, to completely cover the organoids.

    7. Incubate at room temperature for 20 min.

    8. Remove the PFA from the transwells and transfer them to a new wash plate, prepared as in Step A1a, above.

    9. Wash the organoids twice with PBS by repeating Steps A2-A4 as above.

    10. Using a 3 ml plastic Pasteur pipette, take some of the PBS of the last wash in the transwell and expel it onto each organoid, with just enough force so as to dislodge it (but take care not to be too forceful, which can break up the organoid).

    11. Using the same 3 ml Pasteur, transfer each organoid into a separate fresh 1.5 ml Eppendorf tube containing 400 μl PBS. The fixed organoids can be stored at 4°C in PBS for up to one week before embedding.


  2. Embedding fixed tissue in paraffin and sectioning

    A visual guide to the embedding and sectioning of tissues, which is also applicable to our organoids in this section, can be found at this address: https://www.youtube.com/watch?v=7-LIbAWPc-g (accessed in 2021).

    1. Label a number of plastic tissue embedding cassettes (point 8, in Materials and Reagents; Figure 1, Left) equal to the number of organoids with a pencil or permanent marker pen to distinguish between samples.

    2. Place a tissue processing capsule (point 9, in Materials and Reagents; Figure 1, Right) into each plastic tissue cassette, both with lids off.

    3. Using a plastic Pasteur pipette, carefully transfer one organoid into the capsule base.

    4. Replace the lid on the tissue processing capsule, followed by the lid on the tissue cassette.

    5. Immerse the cassette into 70% ethanol.

    6. Repeat Steps B3-B5 for each organoid.

    7. Transfer the cassettes to the tissue processor (Leica ASP300S or equivalent) and run a program following the steps as listed in Table 1.


      Table 1. Tissue processing program (R.T.: room temperature)

    8. Step/Solvent Time Temperature
      70% IMS 20 min R.T.
      70% IMS 30 min R.T.
      90% IMS 45 min R.T.
      90%IMS 60 min R.T.
      100% Absolute Alcohol 30 min R.T.
      100% Absolute Alcohol 45 min R.T.
      100% Absolute Alcohol 60 min R.T.
      Xylene 20 min 40°C
      Xylene 30 min 40°C
      Xylene 40 min 40°C
      Wax 70 min 61°C
      Wax 70 min 61°C
      Wax 70 min 61°C

    9. The next day, take the cassettes out of the tissue processor and transfer them into paraffin wax, using a Heated Paraffin Embedding Station, as follows:

      1. Open the plastic cassette followed by the tissue processing capsule.

      2. With the aid of liquid wax from the embedding station, carefully push the organoid into a metal mold.

      3. Carefully position the organoid in the middle of the mold.

      4. Allow to set for a few seconds on a Cold Plate (see Equipment). Top up the mold with liquid wax, place the plastic cassette, without lid, on the top of the mold and leave it to solidify on the cold plate for about 2 h.

    10. Repeat Steps B8a-B8d for each organoid.

    11. Place the blocks in a container of melting ice/icy water for at least 15 min before sectioning and using a microtome, produce serial 5 µm sections of the paraffin block containing the organoid.

    12. Allow the tissue sections to extend (so that any creases are straightened out) on the surface of clean warm tap water and then transfer them to labelled microscope slides. Allow to dry out on a warm plate and transfer to a 37°C oven overnight for further drying.


  3. Deparaffinization of tissue sections

    Caution: All steps in this section should be performed in a chemical hood.

    Prior to starting the procedure, prepare a series of eleven jars containing the solvent solutions mentioned in each of the steps below.

    1. Place the slides in a slide rack and immerse into a jar of xylene for 5 min.

    2. Immerse in a 2nd jar of xylene for 5 min.

    3. Immerse in a 3rd jar of xylene for 5 min.

    4. Immerse 10 times (2-3 s each time) in a jar of 99.9% ethanol.

    5. Immerse 10 times (2-3 s each time) in a 2nd jar of 99.9% ethanol.

    6. Immerse in a 3rd jar of 99.9% ethanol for 5 min.

    7. Immerse 10 times (2-3 s each time) in a jar of 96% ethanol.

    8. Immerse in a 2nd jar of 96% ethanol for 5 min.

    9. Immerse 10 times (2-3 s each time) in a jar of 70% ethanol.

    10. Immerse in a 2nd jar of 70% ethanol for 5 min.

    11. Immerse in a jar of PBS for 5 min.


  4. Proteinase K treatment

    The Proteinase K concentration and length of treatment presented below were optimized for PFA-fixed, paraffin-embedded human embryonic kidney tissue and kidney organoids. For different fixation methods and/or tissue types, these parameters need to be re-optimized (see Note 2).

    1. Add 0.75 μl Proteinase K stock solution per ml of Proteinase K buffer (see Recipe 5) to obtain 1× Proteinase K reagent. For our fixation/tissue combination (Bantounas et al., 2021), we determined the optimum concentration to be 0.33×; therefore, further dilute the 1× Proteinase reagent 1 in 3, with Proteinase K buffer (see Note 2). Make enough working solution for approximately 300 μl of final reagent per section to be stained (although less may be enough in the case of the organoids).

    2. With the slides on a flat surface, apply 300 μl (or enough to cover the section) of Proteinase K reagent directly onto each section.

    3. Transfer the slides to a pre-heated hybridization oven and incubate at 37°C for 10 min (see Note 2).

    4. Place the slides onto a slide rack and wash twice by immersing into a jar of PBS.


  5. Probe hybridization

    The final probe concentrations and hybridizing temperature given below are specific for the miRNAs we studied in Bantounas et al. (2021) and are given as examples. These parameters should be optimized separately for each individual miRNA (see Note 3). We recommend that alongside the miRNA of interest, a negative control (scrambled) probe, as well as a positive (U6 snRNA) probe is used (see also Table 2).

    1. Dilute the 2× Formamide-free miRNA ISH buffer with an equal volume of RNase-free water to obtain 1× miRNA ISH buffer. Prepare enough for all probe/section combinations (see Step E2).

    2. In an RNase-free Eppendorf tube, dilute the probe stock in 1× miRNA ISH buffer appropriately to obtain the desired working/final concentration LNA® probe mix (see Note 3). Table 2 shows examples of probes optimized at different final concentrations as used in Bantounas et al. (2021). The final volume should be enough to add 50 μl per section (although you can scale up or down depending on the size of your section).


      Table 2. Examples of stock and final concentrations of LNA® probes used on kidney tissue/organoids

    3. Probe Stock concentration Final Concentration Dilution Factor
      U6 snRNA (+ve control) 0.5 μM 0.1 nM 1: 5,000
      miR-199a-3p 25 μΜ 40 nM 1: 625
      miR-214-3p 25 μΜ 1 nM 1: 25,000
      Scrambled (-ve control) 25 μΜ 40 nM 1: 625

    4. Place the tubes in a heating block, at 90°C for 4 min, to denature the probes. Then, centrifuge briefly to collect all the liquid at the bottom of the tube.

    5. Prepare a slide holding staining tray by placing wet tissue at the bottom and place the slides on top, as shown in Figure 2A.

    6. Apply 50 μl of LNA® probe mix onto each section and replace the lid on the staining tray (Figure 2B).



      Figure 2. Set up of slides on staining tray, with wet tissue underneath to prevent evaporation of the applied solution during hybridization


    7. Place the slide box in the oven at 55°C (see Note 3) for 1 h.

    8. Remove the slide box from the oven, place the slides in a slide rack and immerse in a jar containing 5× SSC buffer (Recipe 6).

    9. Prepare a series of six jars containing different concentrations of SSC buffer (see Recipe 6), as detailed below in Table 3, and wash the slides by serially immersing in each of them for 5 min. Keep the jars in a waterbath set at the hybridization temperature, except the last one (#6), which should be at room temperature.


      Table 3. Stringent washes following probe hybridization

    10. Wash # Buffer
      1 5× SSC
      2 1× SSC
      3 1× SSC
      4 0.2× SSC
      5 0.2× SSC
      6 0.2× SSC

    11. Transfer the slides into a jar containing PBS.


  6. Antibody-detection and visualization of bound probe

    Caution: In the blocking and immunostaining steps below, do not let the tissue sections dry out, as this can lead to a very high background signal and also risks compromising the integrity of the tissue.

    1. Using the KP marker (see Materials), apply a hydrophobic barrier around each section.

    2. Add 50 μl (or enough to cover the section) blocking solution (Recipe 8) per section and incubate at room temperature for 15 min.

    3. During blocking, dilute the anti-digoxigenin (anti-DIG-AP) antibody 1:400 (see Note 4) in antibody dilution buffer (Recipe 9), allowing 50 μl (or enough to cover the section) per section.

    4. Remove the blocking solution by tipping the slides, then apply the antibody to the sections and incubate with the antibody at room temperature for 1 h.

    5. Wash the slides three times by placing them in a slide rack and successively immersing in three jars containing fresh PBS-T 0.1% (Recipe 7) for 3 min each.

    6. To make the AP reaction substrate, dissolve an NBT-BCIP tablet in 10 ml distilled water. Then, add 20 μl Levamisol stock solution (see Recipes) (2 μl per ml of NBT-BCIP solution).

      Caution: The substrate solution must be used immediately and be protected from light.

    7. Apply 50 μl (or enough to cover the section) substrate per section and incubate in the dark for 2 h in a 30°C oven (see Note 5).

    8. To stop the reaction, place the slides in a slide rack and immerse in a jar containing KTBT buffer (Recipe 10) for 5 min.

    9. Repeat the above wash a second time.

    10. Wash the slides with distilled water twice, for 1 min each.

    11. Apply 50 μl (or enough to cover the section) Nuclear Fast Red counterstain for 1 min.

    12. Place the slides in a slide rack and into a jar containing tap water. Rinse under running tap water for 10 min.

    13. The slides can be examined and imaged using a brightfield microscope. MiRNA-positive cells will be stained blue. Optionally, to capture the entire section in detail, use a 3D-Histech Panoramic-250 microscope slide-scanner. Subsequently, images can be analysed and snapshots captured with the CaseViewer software and further processed using ImageJ. Representative results can be seen in Figure 3.



      Figure 3. Detection of miRNAs in human fetal kidney sections (week 12 of gestation) by in situ hybridization with digoxigenin-labelled LNA® probes. MiR-199a-3p was present in the stroma and immature glomeruli, while miR-214-3p was present in the stroma, immature glomeruli and tubules (g: glomerulus; t: tubule; str: stroma). Scale bars are 50 μm.

Notes

  1. QIAGEN recommends the use of double-labelled (both 5’ and 3’) digoxigenin probes for miRNA detection. However, we found that in our tissue/miRNA combination, single-labelled (5’) probes gave good results at a lower cost. It is possible that for other tissue/miRNA combinations, a double-labelled probe is indeed necessary (e.g., for less abundant miRNAs).

  2. This protocol assumes PFA fixation and paraffin embedding of tissue sections. If either of these conditions changes, Proteinase K concentration and duration of treatment may need to be re-optimized, even for the same tissue type. In such cases, a pilot experiment must be carried out using the positive control (U6) LNA® probe, varying the range of Proteinase K reagent between 0.03× and 2× and incubating for 10 min. Then, use the concentration that gave the best results and perform a second pilot experiment, varying the duration of treatment between 5 min and 30 min.

  3. The optimal LNA® probe concentration is miRNA-specific, and the optimal hybridization temperature is usually around 30°C below its Tm (as given by the manufacturer). Pilot experiments can be performed for each probe, varying the concentration between 5 nM and 80 nM (although, occasionally, the optimum could be even lower than this; e.g., the miR-214-3p probe in our study was used at 1 nM; Bantounas et al., 2021). The 55°C, mentioned in our protocol, is usually a good starting point as a hybridization temperature, but the optimum can be determined in subsequent pilot experiments, and it usually falls into the 50°C to 60°C range.
     Note: It is possible that no positive results are initially obtained in the probe concentration optimization step, in which case, you should optimize the temperature first using a mid-range probe concentration (e.g., 10-20 nM), following which you should refine the concentration further.

  4. If desired, the antibody dilution can be optimized by using dilutions in the range of 1:200-1:2,000.

  5. If a 2 h development time results in too strong or too weak a signal, you could vary the incubation time of the AP substrate. With some tissue/miRNA combinations, you may be able to observe the colour as it develops under a light microscope and stop or prolong the incubation time accordingly.

Recipes

  1. 4% Paraformaldehyde (PFA) Solution

    1. To a glass beaker, add 800 ml of 1× PBS and heat to 60°C, with stirring on a heated stir plate, in a chemical hood.

    2. Then add 40 g PFA. Raise the pH by adding 10 M NaOH dropwise until the PFA goes into solution (solution becomes clear).

    3. Allow the solution to cool to room temperature and filter (e.g., using a vacuum filter unit).

    4. Adjust the volume to 1,000 ml with 1× PBS.

    5. Check the pH again and adjust with small amounts of 1 M HCl to 6.9.

    6. Store in 10 ml aliquots at -20°C. Defrosted aliquots can be stored at 4°C for up to one month.

  2. 1 M Tris Buffer

    1. In 800 ml distilled water, dissolve 121.1 g Tris Base.

    2. Adjust pH to 7.4 by adding concentrated (37% w/v) HCl (approximately 70 ml).

    3. Make sure the solution is at room temperature before making final adjustments to pH, then adjust the total volume to 1,000 ml before autoclaving. Store indefinitely at room temperature.

  3. 0.5 M EDTA Solution

    1. To 800 ml of distilled water, add 186.1 g disodium-EDTA2H2O.

    2. While stirring using a magnetic stirrer, adjust the pH to 8, using NaOH. Bring volume to 1,000 ml with water.

    3. Autoclave and store at room temperature. Discard if EDTA precipitates out of solution.

  4. 5 M NaCl Solution

    1. Dissolve 292.2 g of NaCl in 800 ml distilled water.

    2. Bring volume to 1,000 ml with water.

    3. Autoclave and store indefinitely at room temperature.

  5. 1× Proteinase K Buffer

    To 900 ml RNase free water, add:

    5 ml of 1 M Tris-HCl, pH 7.4

    2 ml 0.5 M EDTA

    0.2 ml 5 M NaCl

    Bring volume to 1,000 ml, then autoclave. Store indefinitely at room temperature.

  6. SSC Buffer dilutions

    5× SSC: Add 250 ml of 20× SSC to 750 ml distilled water

    1× SSC: Add 50 ml of 20× SSC to 950 ml distilled water

    0.2× SSC: Add 10 ml of 20× SSC to 990 ml distilled water

    Autoclave and store indefinitely at room temperature.

  7. PBS-T 0.1%

    To 1 L of 1× PBS, add 1 ml Tween-20. Store at room temperature for up to one month.

  8. Blocking solution (prepare fresh each time)

    Per 1 ml of PBS-T 0.1% (see Recipe 7 above), add 20 μl sheep serum (2% v/v) and 10 mg BSA (1% w/v). Scale up as necessary.

  9. Antibody dilution buffer (prepare fresh each time)

    1. Prepare PBS-T 0.05% by mixing equal volumes of PBS-T 0.1% (see Recipe 7 above) with PBS.

    2. Then, per 1 ml of the resultant solution, add 10 μl sheep serum (1% v/v) and 10 mg BSA (1% w/v).

  10. KTBT Buffer

    To 900 ml RNase free water, add:

    7.9 g Tris-HCl

    8.7 g NaCl

    0.75 g KCl

    Bring volume to 1,000 ml with water and autoclave. Store indefinitely at room temperature.

Acknowledgments

The protocol is presented as used in the original research paper (Bantounas et al., 2021), in which the probe-binding and visualizing part is an adaptation, with some modifications, of the protocol recommended in QIAGEN’s miRCURY® LNA® miRNA Detection Probes Handbook (October 2017 edition). The following bodies funded the original research: UK Research and Innovation/Medical Research Council (MRC) UK Regenerative Medicine Platform hub Grant MR/K026739/1; Kidney Research UK John Feehally-Stoneygate Project and Innovation award JFS/RP/008/20160916; Kidneys for Life pump priming grant; Horizon 2020 Marie Skłodowska-Curie Actions Initial Training Network RENALTRACT (642937) grant, EPSRC/MRC Centre for Doctoral Training grant EP/L014904/1. We thank Adrian S. Woolf for providing the human embryonic tissue, which was obtained from the MRC and Wellcome Trust Human Developmental Biology Resource. We also thank the University of Manchester Bioimaging Core Facility for helping us capture the images in Figure 3.

Competing interests

The authors declare no competing interests.

Ethics

Human tissues, collected after maternal consent and ethical approval (REC 08/H0906/21+5), were provided by the MRC and Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org/).

References

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  3. Bantounas, I., Ranjzad, P., Tengku, F., Silajdzic, E., Forster, D., Asselin, M. C., Lewis, P., Lennon, R., Plagge, A., Wang, Q., Woolf, A. S. and Kimber, S. J. (2018). Generation of Functioning Nephrons by Implanting Human Pluripotent Stem Cell-Derived Kidney Progenitors. Stem Cell Reports 10(3): 766-779.
  4. Jones, T. F., Bekele, S., O'Dwyer, M. J. and Prowle, J. R. (2018). MicroRNAs in Acute Kidney Injury. Nephron 140(2): 124-128.
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  6. Lopes, F. M., Roberts N. A., Zeef L. A., Gardiner N. J., Woolf A. S. (2019). Overactivity or blockade of transforming growth factor-β each generate a specific ureter malformation. J Pathol 249(4):472-484.
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简介

[摘要] MicroRNAs是负调控基因表达的小RNA,在发育过程中微调分子通路中发挥重要作用。有在肾脏研究它们的功能越来越多的关注,但至今使用肾细胞系多数研究和评估的通过 qPCR 或通过高通量方法(例如下一代测序)确定目标 miRNA 的总量。然而,这几乎没有提供有关 miRNA 在发育中的肾脏中分布的信息,这对于解读它们的作用至关重要,特别是因为有多种肾脏细胞类型,每种类型都有自己特定的转录组。因此,我们提出了一个协议,用于通过肾发育过程中获得用于miRNA表达空间信息原位抗miRNA的杂交(ISH),地高辛标记(DIG),锁核酸(LNA ® )探针上(ⅰ)天然人胚胎组织和 (ii) 人类多能干细胞 (hPSC) 衍生的 3D 肾脏类器官,模拟肾脏发育。我们发现该方法揭示了 miRNA 在特定解剖结构和/或细胞类型中的精确定位,并确认它们在其他细胞中不存在,从而告知它们在发育过程中的特定作用。


[背景] MicroRNA (miRNA) 是小(20 - 25 个核苷酸)RNA,通过主要结合其靶基因 mRNA 的 3' UTR 并抑制其翻译和/或导致其降解来调节基因表达(Bartel ,2018 年)。现在越来越多的研究表明,miRNA 在正常发育和疾病中的肾脏分子通路的微调中发挥着至关重要的作用(Jones等人,2018 年;Trionfini等人,2015 年;Zhao等人, 2019 年)。大多数这些研究是在动物模型上进行的,这些模型并不总是忠实地概括人类的发育事件或疾病表型。为了克服这些限制,我们最近报道了使用人类多能干细胞 (hPSC) 衍生的肾脏类器官(Bantounas等人,2018 年;Takasato等人,2015 年)作为模型来研究miR-199a/214簇的作用在人类肾脏发育过程中(Bantounas等人,2021 年)。在该研究中,我们使用miRNA 与地高辛标记的锁核酸 (LNA ® ) 探针的原位杂交来检测该簇的 miRNA 在天然肾脏和类器官的石蜡切片中的精确定位。LNA ®探针具有化学修饰的骨架,与传统的 DNA 或 RNA 探针相比,每个核苷酸的解链温度 (T m )更高(Singh等,1998)。因此,它们非常适合检测较小的 RNA,常规探针的 T m太低而无法有效结合。结合组织后,将探针检测由碱性磷酸酶(AP)的应用-连接的,抗洋地黄毒苷抗体,然后通过加入AP底物,其被转换为结肠的ü红色产物,可见光下显微镜。使用这种方法,我们发现miR-199a-3p和miR-214-3p都存在于肾基质和发育中的肾小球中,但在成熟的肾小球中基本上不存在。miRNA之一,miR-214-3p ,也表现出强管状表达。此外,我们观察到胚胎/胎儿肾脏和类器官(特别是在miR-199a-3p的情况下)表达程度和分布的差异,可能表明两者代表不同的发育阶段(Bantounas等,2021) . 本示例展示了该方法的主要优势,即获取有关特定组织内感兴趣的 miRNA 表达的空间信息。如果完全依赖转录检测方法,这些信息就会丢失[例如。、qPCR或下一代 miRNA 测序 ( miRseq) ] 。此处介绍的协议的杂交步骤(见下文)改编自 Jørgensen等人。(2010 年)并针对肾脏/肾脏类器官和我们研究的特定 miRNA 进行了优化(Bantounas等人,2021 年)。然而,该方法原则上可用于任何组织的福尔马林固定、石蜡包埋 (FFPE) 切片以及任何 miRNA 或其他类似大小的小 RNA。

关键字:肾脏发育, 类器官, 人类胚胎干细胞, 人胰腺星状细胞, 微小rna, 原位杂交, LNA探针

 
材料和试剂
 
1. 1.5 ml E ppendorf 管(Starlab,目录号:S1615-5550)      
2.塑料移液器吸头(最好过滤)(Starlab,目录号s :S1122-1830 [1,000 μl] ;S1120-8810 [200 μl] ;S1123-1810 [20 μl] ;S1121-3810 [10 μl] ;S1121-3810 [10 μl] )      
3.真空过滤器单元,孔径0.2μm (Thermo,目录号:568-0020)      
4. 6 孔板(Costar,目录号:3516,或等效物)      
5. 3 ml 塑料巴斯德移液器(Starlab,目录号:E1414-0311,或等效物)      
6.组织处理/包埋盒(Simport,目录号:M490-4)(图 1,左)      
7.不锈钢或塑料组织处理胶囊(图 1,右)      
注意:图 1 中描绘的胶囊是作者使用的胶囊,但现已停产。因此,此处提出了替代选项:Simport,目录号:M470;或 Fisher,目录号:15-182-219;或等价物。
 
 
图 1.组织处理盒和胶囊
 
8.用于组织石蜡包埋的金属模具(Leica,目录号:3803081E)      
9.用于工作表面消毒的RNaseZap ®湿巾(Invitrogen/Ambion,目录号:AM9788)      
10. Superfrost ® Plus 载玻片(ThermoFisher Scientific,目录号:J1800AMNZ)   
11.人类胚胎肾组织由 MRC 和 Wellcome Trust Human Developmental Biology Resource ( http://www.hdbr.org/ ) 提供   
12. hPSC 衍生的肾脏类器官可以根据已发表的协议进行生产(Takasato等人,2015 年;Bantounas等人,2018 年和 2021 年)。在我们的研究中(Bantounas et al. , 2021 ),我们分化了MAN13人胚胎干细胞(hESC)系(Ye et al. , 2017 ),但原则上可以使用任何hESC或诱导多能干细胞(iPSC )系。   
13.用于包埋组织的石蜡,熔点61 °C (Pfm Medical,目录号:9000)   
14.无核酸酶水(未经DEPC处理)(ThermoFisher Scientific,目录号:AM9930)   
15.磷酸盐缓冲盐水(PBS),不含Ca 2+和Mg 2+ (Sigma,目录号:D8537-500 ml)   
16. 5'-地高辛标记的 miRCURY ® LNA ® miRNA 检测探针(1 nmol)(见注 1 )(QIAGEN,目录号:339111;见下文了解单个探针代码)。在这个例子中,我们使用了探针定位:   
miR-199a-3p (QIAGEN,目录号:YD00615410)
miR-214-3p (QIAGEN,目录号:YD00611471)
乱序探针(阴性对照;QIAGEN,目录号:YD00699004)
U6 snRNA(阳性/优化对照;QIAGEN,目录号:YD00699002)
17. miRCURY ® LNA ®的miRNA ISH缓冲器组(FFPE)(QIAGEN,目录号:339450),其中包括:   
2 × 无甲酰胺 miRNA ISH 缓冲液
蛋白酶K溶液
18. UltraPure TM 20 × SSC 缓冲液(Invitrogen,目录号:15557-044)   
19. Tween-20(Sigma,目录号:P1379-250ML)   
20.绵羊抗DIG-AP(碱性磷酸酶连接)抗体(Roche/Sigma-Aldrich,目录号:11093274910)   
21.羊血清(Sigma-Aldrich,目录号:S3772-10ML)   
22.牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A1470-100G)   
23.硝基蓝四唑/5-溴-4-氯-吲哚基-磷酸(NBT/BCIP)即用型片剂(Roche,目录号:1 1 697 471 001)   
24.左旋咪唑(Fluka,目录号:31742),用水稀释制成 100 mM 储备溶液   
25.核固红溶液(默克,目录号:N3020)   
26.工业甲基化酒精(IMS)(各种供应商)   
27.二甲苯(各种供应商)   
28. 100% 乙醇(各种供应商)   
29. KCl (各种供应商)   
30. KP Marker Plus(Histolab,目录号:98307-R)或类似的疏水标记笔(各种供应商)   
31. 4% 多聚甲醛 (PFA) 溶液(见配方)   
32. 1 M Tris 缓冲液(见配方)   
33. 0.5 M EDTA 溶液(见配方)   
34. 5 M NaCl 溶液(见配方)   
35. 1 ×蛋白酶 K 缓冲液(见配方)   
36. SSC 缓冲液稀释(见配方)   
37. PBS-T 0.1%(见食谱)   
38.阻塞解决方案(见食谱)   
39.抗体稀释缓冲液(见配方)   
40. KTBT 缓冲液(见食谱)   
 
设备
 
组织处理器(徕卡,型号:ASP300S ;或同等产品)
加热石蜡包埋站(Leica HistoCore Arcadia H 或同等产品)
冷板(Leica、HistoCore Arcadia C 或同等产品)
切片机(徕卡,型号:RM225 ;或同等产品)
杂交炉或其它可变温度培养箱(例如。,的Hybaid,南部双14或等同物)
显微镜载玻片架(Simport,目录号:M905-12DGY)
用于将载玻片架浸入溶剂/缓冲液中的罐子(Simport,目录号:M900-12G)
加热块(最高 90 °C )(Eppendorf ThermoMixer F1.5 或同等产品)
微量离心机(Labnet、Prism 或同等产品)
Gilson ®或等效移液器(各种供应商)
用于在杂交和染色过程中固定载玻片的染色托盘(见图 2 )。
注意:图 2 中描绘的托盘是作者使用的托盘,但现已停产。此处提出了替代方案:Fis her,目录号:22-045-035 。
水浴(Grant SUB6,目录号:P266 ;或等效物)
高压釜(各种供应商)
可选的成像:3D-Histech Panoramic-250 显微镜载玻片扫描仪,带有 40 × /0.95 Plan Apochromat 物镜(蔡司)
 
软件
 
可选:CaseViewer(3DHISTECH Ltd.;www.3dhitech.com),在使用 3D-Histech 幻灯片扫描仪扫描后捕获图像(参见设备中的第 14 点)
斐济/ImageJ ( http://imagej.net/Fiji/Downloads ) (用于捕获后的图像处理)
 
程序
 
组织固定
本节中描述的程序用于制备类器官组织,是对 Lopes等人中描述的程序的改编。(2019 年)。
如果从已经包埋在石蜡中的样品开始:直接跳到 B 部分,步骤 10。
以下程序假设在 6 孔板的 transwell 插入物中培养类器官(Bantounas等人,2018 年)。如果使用不同大小井和/或插入时,变化成比例地卷在小号TEPS甲1-甲7。
准备“洗涤”和“固定”6 孔板,允许每个 transwell 插入一个孔有机体:
洗板:在每个孔中加入 1.2 ml PBS 。
固定板:在每个孔中加入 1.2 ml 4% PFA 。
将 transwell 插入物从培养板转移到上面准备好的洗涤板。
小心地将 1 ml PBS 添加到每个 transwell 中并轻轻旋转板以洗涤有机体。
使用 Gilson 或 3 ml Pasteur 移液器,从 transwell 内取出 PBS。
从“洗”传送转孔的“固定”板(即。,在PFA已在每个孔中的顶部)。
将 1 ml 4% PFA 添加到每个 transwell 中,确保类器官被完全覆盖。
注意:如有必要,您可以添加更多 PFA以完全覆盖有机体。
在室温下孵育 20 分钟。
从转孔移除PFA并将它们转移到一个新的洗涤板,制备成在小号TEP甲1a中,以上。
通过重复用PBS洗两次类器官小号TEPS甲2-甲4如上述。
使用 3 ml 塑料巴斯德移液管,在 transwell 中取出一些最后一次洗涤的 PBS 并将其排出到每个类器官上,力道刚好足以将其移开(但注意不要用力过猛,否则会破裂)类器官)。
使用相同的 3 ml Pasteur,将每个有机体转移到一个单独的新鲜 1.5 ml Eppendorf 管中,其中含有 400 μl PBS。固定的有机体可以在 PBS中 4 °C下储存长达一周,然后再嵌入。
 
在石蜡中嵌入固定组织并切片
组织嵌入和切片的视觉指南,也适用于本节中的类器官,可在以下地址找到:https : //www.youtube.com/watch?v=7- LIbAWPc-g (已访问) 2021 年)。
标注号塑料组织包埋盒(点8 ,在材料和- [R eagents ;图1,左)等于用铅笔或永久性标记笔样品之间进行区分类器官的数量。
放置一个组织处理胶囊(点9 ,在材料和- [R eagents ;图1,右)到每个塑料组织盒,都与关闭盖子。
使用塑料巴斯德吸管,小心地将一个类器官转移到胶囊底座中。
更换组织处理胶囊上的盖子,然后更换组织盒上的盖子。
将卡带浸入 70% 乙醇中。
重复小号TEPS乙3-乙5为每个类器官。
将卡带转移到组织处理器(Leica ASP300S 或同等产品)并按照表 1 中列出的步骤运行程序。
 
表 1. 组织处理程序(R . T . :室温)
 
第二天,将包埋盒从组织处理器中取出,使用加热石蜡包埋台将它们转移到石蜡中,如下所示:
打开塑料盒,然后是组织处理胶囊。
借助来自嵌入站的液体蜡,小心地将有机体推入金属模具中。
小心地将有机体放置在模具的中间。
允许在冷板上放置几秒钟(参见设备)。用液态蜡加满模具,将没有盖子的塑料盒放在模具顶部,让它在冷板上凝固约 2 小时。
重复小号TEPS乙8A- B8 d为每个类器官。
在切片和使用切片机之前,将块s 放入融化的冰/冰水的容器中至少 15 分钟,生成包含有机体的石蜡块的连续 5 μm 部分。
让组织切片在干净温暖的自来水表面上延伸(使任何折痕变直),然后将它们转移到标记的显微镜载玻片上。允许在温暖的盘子上晾干,并在一夜之间转移到 37 °C 的烤箱中进一步干燥。
 
组织切片的脱蜡
注意:本节中的所有步骤都应在化学罩中进行。
在开始该程序之前,准备一系列包含以下每个步骤中提到的溶剂溶液的 11 个罐子。
将载玻片放在载玻片架中,然后浸入一罐二甲苯中 5 分钟。
浸入第二个二甲苯罐中 5 分钟。
浸入第三个二甲苯罐中 5 分钟。
在装有 99.9% 乙醇的罐子中浸泡 10 次(每次2-3秒)。
在第二个装有99.9% 乙醇的罐子中浸泡 10 次(每次 2-3 秒)。
沉浸在3次99.9%的乙醇5分钟罐子。
在 96% 乙醇的罐子中浸泡 10 次(每次 2-3 秒)。
浸入第二罐 96% 乙醇中 5 分钟。
在装有 70% 乙醇的罐子中浸泡 10 次(每次2-3 秒)。
浸入第二罐 70% 乙醇中 5 分钟。
沉浸在一个PBS的5分钟罐子。
 
蛋白酶K处理
下面介绍的蛋白酶 K 浓度和处理时间针对 PFA 固定、石蜡包埋的人胚胎肾组织和肾类器官进行了优化。对于不同的固定方法和/或组织类型小号,这些参数需要被重新优化(见注2 )。
每毫升蛋白酶 K 缓冲液(参见配方 5 )加入 0.75 μl 蛋白酶 K 储备溶液以获得 1 ×蛋白酶 K 试剂。(Bantounas对于我们的固定/组织组合等。,2021),我们确定了最佳浓度为0.33 × ; 因此,用蛋白酶 K 缓冲液进一步稀释 1 ×蛋白酶试剂 1:3(见注 2 )。为每个要染色的切片制作大约 300 μl 的最终试剂的足够工作溶液(尽管在有机体的情况下,更少可能就足够了)。
将载玻片放在平坦的表面上,将 300 μl(或足以覆盖切片)的蛋白酶 K 试剂直接涂在每个切片上。
将载玻片转移到预热的杂交炉中,并在 37 °C下孵育10 分钟(见注 2 )。
将载玻片放在载玻片架上,浸入一罐 PBS 中洗涤两次。
 
探针杂交
下面给出的最终探针浓度和杂交温度特定于我们在 Bantounas等人中研究的 miRNA 。( 2021 )并作为示例给出。这些参数应该针对每个单独的 miRNA 单独优化(参见注释 3 )。我们建议在感兴趣的 miRNA 旁边,使用阴性对照(乱序)探针,以及阳性(U6 snRNA)探针(另见表 2 )。
用等体积的无 RNase 水稀释2 × Formamide-free miRNA ISH 缓冲液,以获得 1 × miRNA ISH 缓冲液。对于所有探针/部分的组合(见足够准备小号TEP ë 2)。
在无 RNase 的E ppendorf 管中,在 1 × miRNA ISH 缓冲液中适当稀释探针储备,以获得所需的工作/最终浓度 LNA ®探针混合物(参见注释 3 )。表 2 显示了在 Bantounas等人中使用的在不同最终浓度下优化的探针示例。(2021 年)。最终体积应该足以为每个切片添加 50 μl (尽管您可以根据切片的大小放大或缩小)。
 
表 2.用于肾脏组织/类器官的 LNA ®探针的储备和最终浓度示例
 
将管子放在加热块中,在 90 °C 下加热4 分钟,使探针变性。然后,短暂离心以收集管底部的所有液体。
通过在底部放置湿纸巾并将幻灯片放在顶部来准备一个幻灯片固定染色托盘,如图 2A所示。
将 50 μl LNA ®探针混合物涂抹在每个切片上,并盖上染色托盘的盖子(图 2B )。
 
             
图 2.在染色托盘上设置载玻片,下面有湿组织,以防止在杂交过程中应用的溶液蒸发
 
将滑动盒放入 55 °C的烤箱中(见注 3 )1 小时。
从烤箱中取出载玻片盒,将载玻片放入载玻片架并浸入含有 5 × SSC 缓冲液的罐子中(配方 6 )。
制备一系列的含有不同浓度的六缸的SSC缓冲液(见配方6 ),如下面详述在表3中,并且通过在它们中的每连续浸渍5分钟洗滑动。将罐子放在设置为杂交温度的水浴中,最后一个 (#6) 除外,它应该在室温下。
 
表3 。探针杂交后的严格洗涤
 
将载玻片转移到装有 PBS 的罐子中
 
结合探针的抗体检测和可视化
注意:在下面的封闭和免疫染色步骤中,不要让组织切片变干,因为这会导致非常高的背景信号,也有损害组织完整性的风险。
使用 KP 标记(参见材料),在每个部分周围应用疏水屏障
每切片加入 50 μl(或足以覆盖切片)封闭液(配方 8 ),并在室温下孵育 15 分钟。
在封闭过程中,在抗体稀释缓冲液(配方 9 )中以1:400(见注 4 )稀释抗地高辛(抗 DIG-AP)抗体,每个切片允许 50 μl(或足以覆盖切片)。
通过倾斜载玻片去除封闭溶液,然后将抗体应用于切片,并在室温下与抗体一起孵育 1 小时。
将m放在载玻片架上,依次浸入三个装有新鲜 PBS-T 0.1%(配方 7 )的罐子中,每次 3 分钟,将载玻片清洗 3 次。
要制备 AP 反应底物,请将n NBT-BCIP 片剂溶解在 10 ml 蒸馏水中。然后,添加 20 μl 左旋咪唑原液(参见配方)(每毫升 NBT-BCIP 溶液 2 μl)。
注意:底物溶液必须立即使用并避光。
每部分应用 50 μl(或足以覆盖该部分)底物,并在 30 °C烤箱中在黑暗中孵育 2 小时(见注 5 )。
要停止反应,请将载玻片放在载玻片架中,然后浸入含有 KTBT 缓冲液(配方 10 )的罐子中5 分钟。
重复上述洗涤第二次。
用蒸馏水清洗载玻片两次,每次 1 分钟。
应用 50 μl(或足以覆盖切片)核固红复染剂 1 分钟。
将载玻片放入载玻片架并放入盛有自来水的罐子中。在流动的自来水下冲洗 10 分钟。
可以使用明场显微镜检查和成像载玻片。miRNA 阳性细胞将被染成蓝色。或者,要详细捕获整个部分,请使用3D-Histech Panoramic-250 显微镜载玻片扫描仪。随后,可以使用CaseViewer 软件分析图像和捕获快照,并使用 ImageJ 进一步处理。代表性的结果可以在图 3 中看到。
 
 
图 3. 通过与地高辛标记的 LNA ®探针原位杂交检测人胎儿肾脏切片(妊娠第 12 周)中的 miRNA 。MiR-199a-3p存在于基质和未成熟肾小球中,而miR-214-3p存在于基质、未成熟肾小球和肾小管(g:肾小球;t:肾小管;str:基质)。比例尺为 50 μm。
 
笔记
 
QIAGEN推荐小号用于miRNA检测使用双标记的(5'和3' )地高辛探针。然而,我们发现在我们的组织/miRNA 组合中,单标记 (5') 探针以较低的成本提供了良好的结果。可能对于其他组织/miRNA 组合,双标记探针确实是必要的(例如,对于较少丰度的miRNA)。
该协议假定组织切片的 PFA 固定和石蜡包埋。如果这些条件中的任何一个发生变化,蛋白酶 K 浓度和治疗持续时间可能需要重新优化,即使对于相同的组织类型也是如此。在这种情况下小号,导频实验必须使用阳性对照(U6)LNA进行®探针,改变蛋白酶K试剂的范围0.03之间×和2 ×并孵育10分钟。然后,使用给出最佳结果的浓度并进行第二次试点实验,在 5分钟到 30 分钟之间改变治疗持续时间。
最佳的 LNA ®探针浓度是 miRNA 特异性的,最佳杂交温度通常比其 T m (由制造商提供)低30 °C左右。可以对每个探针进行先导实验,在 5 nM 和 80 nM 之间改变浓度(尽管有时最佳浓度甚至可能低于此值;例如,我们研究中使用的miR-214-3p探针为 1 nM; Bantounas等人。,2021)。我们的协议中提到的55 °C通常是作为杂交温度的一个很好的起点,但可以在后续的试点实验中确定最佳值,并且通常落在 50 °C到 60 °C 的范围内。Ñ OTE:即,能够无阳性结果列于探针浓度优化步骤最初获得,在这种情况下,应该首先使用中档探针浓度(例如优化的温度,10-20纳米),随后你应该进一步细化浓度。
如果需要,可以使用 1:200-1:2 , 000范围内的稀释度来优化抗体稀释度。
如果 2小时的开发时间导致信号太强或太弱,您可以改变 AP 底物的孵育时间。对于某些组织/miRNA 组合,您可以在光学显微镜下观察颜色的变化,并相应地停止或延长孵育时间。
 
食谱
 
4% 多聚甲醛 (PFA) 溶液
向玻璃烧杯中加入 800 ml 1× PBS 并加热至 60°C,同时在加热的搅拌板上,在化学罩中搅拌。
然后加入 40 克粉煤灰。通过滴加 10 M NaOH 来提高 pH 值,直到 PFA 进入溶液(溶液变得清澈)。
让溶液冷却至室温并过滤(例如,使用真空过滤装置)。
用 1 × PBS将体积调节至 1,000 ml 。
再次检查 pH 值并用少量 1 M HCl 调节至 6.9。
在 -20°C 下以 10 ml 等分试样储存。解冻的等分试样可在 4°C 下储存长达 1 个月。
1 M Tris 缓冲液
在 800 毫升蒸馏水中,溶解 121.1 克 Tris 碱。
通过添加浓 (37% w/v) HCl (约 70 ml) 将 pH 值调节至 7.4。
在最终调整 pH 值之前确保溶液处于室温,然后在高压灭菌前将总体积调整为 1,000 毫升。在室温下无限期储存。
0.5 M EDTA 溶液
向 800 ml 蒸馏水中加入 186.1 g 二钠-EDTA∙2H 2 O。
在使用磁力搅拌器搅拌的同时,使用 NaOH 将 pH 值调整为 8。用水定容至 1,000 毫升。
高压灭菌并在室温下储存。如果 EDTA 从溶液中沉淀出来,则丢弃。
5 M 氯化钠溶液
将 292.2 g NaCl 溶解在 800 ml 蒸馏水中。
用水定容至 1,000 毫升。
高压灭菌并在室温下无限期储存。
1×蛋白酶K缓冲液
900毫升不含Rnase水,加入:
5毫升的1的1M Tris-HCL,pH为7.4
2 毫升 0.5 M EDTA
0.2 毫升 5 M 氯化钠
使体积达到 1,000 毫升,然后高压灭菌。在室温下无限期储存。
SSC 缓冲液稀释
5 × SSC:将 250 ml 20 × SSC添加到 750 ml 蒸馏水中
1 × SSC:将 50 ml 20 × SSC添加到 950 ml 蒸馏水中
0.2 × SSC:将 10 ml 20 × SSC添加到 990 ml 蒸馏水中
高压灭菌并在室温下无限期储存。
PBS-T 0.1%
向 1 L 1 × PBS 中加入 1 ml Tween-20。在室温下最多可存放 1 个月。
封闭溶液(p每次repare新鲜)
每 1 ml PBS-T 0.1%(参见上述配方 7 ),添加 20 μl 绵羊血清(2% v/v)和 10 mg BSA(1% w/v)。根据需要扩大规模。
抗体稀释缓冲液(p每次repare新鲜)
将等体积的 PBS-T 0.1%(参见上述配方 7 )与 PBS混合,制备 PBS-T 0.05% 。
然后,每 1 ml 所得溶液加入 10 μl 羊血清(1% v/v)和 10 mg BSA(1% w/v)。
KTBT 缓冲器
900毫升不含Rnase水,加入:
7.9 克 Tris-HCl
8.7 克氯化钠
0.75 克氯化钾
用水和高压釜使体积达到 1,000 毫升。在室温下无限期储存。
 
致谢
 
该协议在原始研究论文(Bantounas等人,2021 年)中使用,其中探针结合和可视化部分是对 QIAGEN 的 miRCURY ® LNA ® miRNA 检测中推荐的协议的改编和一些修改探针手册(2017 年 10 月版)。以下机构资助了原始研究:英国研究与创新/医学研究委员会 (MRC) 英国再生医学平台中心 Grant MR/K026739/1;英国肾脏研究 John Feehally-Stoneygate 项目和创新奖 JFS/RP/008/20160916;生命肾脏泵启动补助金;地平线 2020 Marie Skłodowska - Curie Actions 初始培训网络 RENALTRACT (642937) 赠款,EPSRC/MRC 博士培训中心赠款 EP/L014904/1。我们感谢 Adrian S. Woolf 提供了从MRC 和 Wellcome Trust 人类发育生物学资源获得的人类胚胎组织。我们也感谢曼彻斯特大学的生物成像中心实验室帮助我们捕捉到的图像˚F igure 3。
 
利益争夺
 
作者声明没有竞争利益。
 
伦理
 
在母亲同意和伦理批准 (REC 08/H0906/21+5) 后收集的人体组织由MRC 和 Wellcome Trust 人类发育生物学资源 ( http://www.hdbr.org/ ) 提供。
 
参考
 
巴特尔,DP (2018)。后生动物微小RNA。单元格173(1):20-51。
Bantounas, I., Lopes, FM, Rooney, KM, Woolf, AS 和 Kimber, SJ (2021)。miR-199a/214 簇控制人胚胎干细胞模型中的肾形成和血管化。干细胞报告16(1): 134-148。
Bantounas, I., Ranjzad, P., Tengku, F., Silajdzic, E., Forster, D., Asselin, MC, Lewis, P., Lennon, R., Plagge, A., Wang, Q., Woolf , AS 和 Kimber, SJ (2018)。通过植入人多能干细胞衍生的肾祖细胞产生功能性肾单位。干细胞报告10(3): 766-779。
Jones, TF、Bekele, S.、O'Dwyer、MJ 和 Prowle, JR(2018 年)。急性肾损伤中的微小RNA。肾单位140(2):124-128。
Jørgensen S.、Baker A.、Møller S.、Nielsen BS (2010)。使用 LNA 探针检测石蜡样品中 microRNA 的可靠一日原位杂交方案。方法52(4):375-81。
Lopes , FM, Roberts NA, Zeef LA, Gardiner NJ, Woolf AS (2019)。转化生长因子-β 的过度活跃或阻断都会导致特定的输尿管畸形。 J Pathol 249(4):472-484。
Singh SK、Nielsen P.、Koshkin AA 和 Wengel J. (1998)。LNA(锁核酸):合成和高亲和力酸识别。化学通讯455-456。
Takasato, M., Er, PX, Chiu, HS, Maier, B., Baillie, GJ, Ferguson, C., Parton, RG, Wolvetang, EJ, Roost, MS, Chuva de Sousa Lopes, SM and Little, MH ( 2015)。来自人类 iPS 细胞的肾脏类器官包含多个谱系和人类肾发生模型。自然526(7574):564-568。
Trionfini, P.、Benigni, A. 和 Remuzzi, G. (2015)。MicroRNAs 在肾脏生理和疾病中的作用。Nat Rev Nephrol 11(1): 23-33。
Ye, J., Bates, N., Soteriou, D., Grady, L., Edmond, C., Ross, A., Kerby, A., Lewis, PA, Adeniyi, T., Wright, R., Poulton , KV, Lowe, M., Kimber, SJ 和 Brison, DR (2017)。来自新鲜废弃胚胎的高质量临床级人类胚胎干细胞系。干细胞水疗8(1): 128。
Zhao, H.、Ma, SX、Shang、YQ、Zhang、HQ 和 Su, W.(2019 年)。慢性肾病中的微小RNA。Clin Chim Acta 491:59-65。
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引用:Lopes, F. M., Kimber, S. J. and Bantounas, I. (2021). In situ Hybridization of miRNAs in Human Embryonic Kidney and Human Pluripotent Stem Cell-derived Kidney Organoids. Bio-protocol 11(17): e4150. DOI: 10.21769/BioProtoc.4150.
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