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Feb 2018

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A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol
一种广泛应用的尿素荧光/比色mrna原位杂交技术   

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Abstract

In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence–the hybridization step, followed by detection of annealed sequences by means of fluorescent or colorimetric reactions. Successful hybridization requires permeabilization of tissues, followed by denaturation of nucleic acids strands, which is usually carried out in a formamide-based buffer and at high temperatures. Such reaction conditions, besides posing a health hazard (both concerning manipulation and waste disposal), can be excessively harsh for the delicate tissues of some species or developmental stages. We detail here an alternative method for in situ hybridization, where the toxic formamide is replaced with a urea solution. This substitution improved both tissues preservation and signal-to-noise detection, in several animal species. The protocol described here, originally developed for the hydrozoan jellyfish Clytia hemisphaerica, provides guidelines for adapting formamide-based traditional protocols to the urea variant. Urea-based protocols have already been successfully applied to diverse invertebrate and vertebrate species, showing the ease of such a modification, and providing the scientific community with a promising, safer and versatile tool.

Keywords: mRNA in situ hybridization (RNA原位杂交), Urea (尿素), Formamide (甲酰胺), Health and safety (健康和安全), Signal-to-noise (信噪比)

Background

The pairwise complementary binding of single strand nucleic acid sequences inspired the development of a powerful technique, in situ hybridization, which allows researchers to visualize the location of DNA or RNA strands within their cellular and tissue contexts (Pardue and Gall, 1969). Numerous variants have since been developed, differing for the labeling of the exogenous complementary probes (e.g., radioactive or hapten-based), in the type of samples (e.g., for whole embryos or tissue sections) and in the detection method (e.g., fluorescent or colorimetric signals).

Precise annealing of probes depends on the accessibility of the target sequences, achieved through appropriate permeabilization of tissues and cell membranes (which allows penetration of reagents), and denaturation of nucleic acid strands (which exposes the complementary target sequences). Achieving adequate sensitivity of detection, specificity of annealing, and preservation of sample morphology often requires a time-consuming optimization of reaction conditions. Efficient denaturation and annealing of nucleic acids strands are usually obtained by means of elevated temperatures and the addition of a denaturing reagent in the hybridization buffer.

Reaction temperatures should ideally be adapted to the base composition of the sequences of interest: hybridization temperatures are about 25 °C below denaturation temperatures (Tm; Marmur and Doty, 1961). 50-70% formamide is usually employed as the denaturing reagent, this choice remaining largely unchallenged since the ‘80s–with few exceptions, such as the smFISH (single-molecule Fluorescence In Situ Hybridization) approach where 10-15% formamide is used (Haimovich and Gerst, 2018).

Elevated temperatures increase evaporation of the hybridization buffer, compounding the health risk already posed by formamide, an irritating, embryotoxic and teratogenic solvent (Gleich, 1974; Stula and Krauss, 1977; Merkle and Zeller, 1980; Kennedy and Short, 1986; Fail et al., 1998; George et al., 2000 and 2002; see also Sinigaglia et al., 2018 for further information). Hybridizing samples and waste need to be appropriately handled (CICAD 31), thus limiting the application of standard formamide-based protocols in sensitive contexts, such as pregnancy or in classrooms.

In the early years, a diversity of solvents was found to effectively destabilize nucleic acid strands, with urea being particularly efficient (Herskovits, 1963). Indeed, urea and formamide have similar properties, and are employed equivalently in a number of applications, such as fluorescent in situ hybridization on bacteria (Fontenete et al., 2016), protein denaturation, and clearing agents for imaging (reviewed in Azaripour et al., 2016). Urea can effectively replace formamide in Northern and Southern blot experiments, in particular at concentrations in the 2 M-4 M range (Simard et al., 2001). The mechanism of action of urea is still relatively poorly understood. Urea lowers the Tm of DNA of about 2 °C per mole of urea (Hutton, 1977), and is thus slightly less efficient than formamide, which achieves a 2.4-2.9 °C reduction per mole. Urea molecules bind weakly to RNA, disrupting its base-pair interactions and ultimately destabilizing the structure of RNA molecules (Herskovits and Bowen, 1974; Priyakumar et al., 2009; Lambert and Draper, 2012).

Urea, in powder or crystal form, is water soluble, and 8 M stock solutions can be readily prepared at 20 °C. Solutions tend to be viscous (Hutton, 1977), which might explain why high concentrations of urea are less effective (Simard et al., 2001). Such increased viscosity demands care during the hybridization step of the in situ protocol, to ensure that samples are efficiently immersed, and evaporation minimized.

The urea-based protocol presented here has been shown to be more efficient than the standard formamide-based ones in diverse metazoan species and at different developmental stages (see Sinigaglia et al., 2018). Detection sensitivity was improved, allowing visualization of unsuspectedly complex patterns of gene expression (Sinigaglia et al., 2018). This increased sensitivity of detection might be explained by an additional permeabilizing action of urea on tissues (Lim et al., 2009; Huang et al., 2011), likely due to increased osmotic pressure within cells (reviewed in Tainaka et al., 2016). The hyperhydration of tissues might also partially account for the improved morphologies of delicate samples, shown in the original research paper (Sinigaglia et al., 2018).

The protocol detailed here was originally developed for the hydrozoan medusa Clytia hemisphaerica (Quiroga Artigas et al., 2018; Sinigaglia et al., 2018, Leclère et al., 2019). Following those guidelines, urea-alternative protocols have already been developed for a diversity of metazoan species and developmental stages, including priapulid and brachiozoan embryos (Thiel et al., 2017; Sinigaglia et al., 2018), the acoel Hofstenia miamia (L. Ricci, personal communication), the scyphozoan jellyfish Aurelia aurita (M. Manuel and T. Condamine, personal communication), and in the paddlefish (M. Minarik, personal communication). Substituting formamide with urea has also been applied to DNA-FISH on mouse oocytes (Manil-Segalen et al., 2018), further showing the versatility of this approach.

Such versatility stems from the simplicity of the key implementation, the substitution of formamide with an equal volume of urea solution (at a final concentration of 4 M). When adapting previous protocol to novel species or tissues, it is recommended to start by simply switching formamide to urea, leaving the rest of the procedure unchanged. Guidelines for eventual troubleshooting are also provided, offering general recommendations for successful in situ hybridization.

Materials and Reagents

  1. Materials and reagents used in multiple steps
    1. Gloves
    2. RNase free tubes (e.g., 1.5 ml Eppendorf tubes, or equivalent)
    3. In situ baskets (Sample container, as in Figure 1A)
      They could be either the Netwell baskets (Corning), or homemade baskets constructed from nylon mesh and microcentrifuge tubes, as explained in Sive et al. (2007). Baskets size depends on the type of sample; they usually fit within the wells of a 6-, 12- or 24-well plate. Performing in situ hybridization in baskets (instead of tubes) is recommended: 
      1. To reduce loss and damages to samples, due to pipetting.
      2. To accelerate exchange of solutions (baskets can be simply transferred with the help of forceps to a multiwell plate with the new solution (Figure 1B)–in this case tap gently the basket against the walls of the well to ensure removal of older solution, then gently immerse it in the new well).
      3. Baskets can be recycled multiple times, after being thoroughly washed (e.g., rinse with MilliQ H2O, then immerse in a 1 M NaOH solution to neutralize RNases, and rinse several times with RNase-free MilliQ H2O–simply placing them in a beaker on a shaker will assure thorough swirling).


        Figure 1. Sample carrier. A. Example of three baskets with mesh-bottom. B. A 24-well plate used for in situ hybridization, with four baskets. C. The same plate, closed and wrapped in plastic film to avoid evaporation during hybridization step.

    4. Multiwell plate (For fitting in situ baskets, as in Figure 1B); (e.g., 24-well cell culture plate, sterile, with lid, from CellStar (Greiner Bio-one, catalog number: 662-160))
    5. Ice and ice container
    6. MilliQ H2O (Storage: RT)
    7. Tween-20 (Sigma-Aldrich, catalog number: P1379), storage: RT (Mild detergent. Given its high viscosity, it is recommended to prepare a 10% working solution)
    8. Na2HPO4·7H2O (Sigma-Aldrich, catalog number: 431478), storage: RT (For preparing 10X PBS solution)
    9. NaCl (Sigma-Aldrich, catalog number: S9888), storage: RT (For preparing 10X PBS solution)
    10. KCl (Sigma-Aldrich, catalog number: P9333), storage: RT (For preparing 10X PBS solution)
    11. KH2PO4 (Sigma-Aldrich, catalog number: P9791), storage: RT (For preparing 10X PBS solution)
    12. 10x PBS (Phosphate-Buffered Saline) pH 7.4 (see Recipes, or from tablets from Calbiochem, catalog number: 524650), storage: RT (Helps maintaining a constant pH)

  2. Fixation
    1. 37% formaldehyde (Sigma-Aldrich, catalog number: 252549), storage: RT ( Crosslinking)
    2. 8% glutaraldehyde (Electron Microscopy Sciences, catalog number: 16020 ), storage: 4 °C ( Crosslinking)
    3. EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid) (Sigma-Aldrich, catalog number: E3889), storage: RT [Calcium/magnesium chelator, which reduces enzyme activity (such as RNase); reagent of HEM buffer]
    4. HEPES (Sigma-Aldrich, catalog number: H4034), storage: RT (Reagent of HEM buffer)
    5. MgSO4 (Sigma-Aldrich, catalog number: M7506), storage: RT (Reagent of HEM buffer)
    6. Tween-20 (Sigma-Aldrich, catalog number: P1379), storage: RT
    7. Methanol (e.g., Sigma-Aldrich, catalog number: 322415), storage: RT
    8. 1x PBS (see Recipes)
    9. 1x PBST (see Recipes)
    10. CISH fixation buffer (see Recipes)
    11. FISH fixation buffer (see Recipes)
    12. Dehydration buffer (see Recipes)
    13. Rehydration buffer (see Recipes)
    14. Re-fixation buffer (see Recipes)

  3. Acetylation
    1. TEA (Triethylamine) (Sigma-Aldrich, catalog number: T0886), storage: RT (For reducing the non-specific binding of negatively-charged probe to tissues)
    2. Acetic anhydride (Sigma-Aldrich, catalog number: A6404), storage: 4 °C (For reducing the non-specific binding of negatively-charged probe to tissues)
    3. 1x PBST (see Recipes)
    4. 10x TEA stock solution (see Recipes)
    5. 0.1 M TEA (see Recipes)
    6. 0.25% Acetic anhydride in 0.1 M TEA (see Recipes)

  4. Permeabilization
    1. Proteinase K (Sigma-Aldrich, catalog number: P4850), storage: 4 °C or -20 °C (Digests proteins, including nucleases. Optimum pH is 7.5-9.0)
    2. Glycine (Sigma-Aldrich, catalog number: G8898), storage: RT (For neutralizing the proteinase K)
    3. 1x PBST (see Recipes)

  5. Hybridization
    1. Plastic film (For wrapping sample container during hybridization step)
    2. Urea (Sigma-Aldrich, catalog number: U5378), storage: RT (Denaturing agent)
    3. Dextran sulfate sodium salt, Mr ~200''00 (Sigma-Aldrich, catalog number: 67578-25G), storage: RT [Increases molecular crowding and can therefore accelerate the kinetics of hybridization (locally increases probe concentration)]
    4. tRNA, from baker’s yeast (Sigma-Aldrich, catalog number: 10109509001), storage: -20 °C (Blocks non-specific probe binding)
    5. Salmon sperm (Thermo Fisher, catalog number: 15632011), storage: -20 °C (Blocks non-specific probe binding)
    6. Heparin sodium (Thermo Fisher, catalog number: 10239840), storage: -20 °C (Helps to reduce background, by binding to proteins that bind to DNA)
    7. 20% SDS (Sodium Dodecyl Sulfate) (Fisher, catalog number: BP131), storage: RT (Detergent. Permeabilizes membranes, facilitating probe distribution)
    8. NaCl (Sigma-Aldrich, catalog number: S9888), storage: RT (Salt. For preparing 20x SSC stock buffer, see Recipes)
    9. Trisodium citrate (Na3C6H5O7) (For preparing 20x SSC stock buffer, see Recipes)
    10. Tween-20 (Sigma-Aldrich, catalog number: P1379), storage: RT
    11. 10x DIG labeling mix RNA (Roche, catalog number: 11277073910), storage: -20 °C (For probe synthesis)
    12. RNasin Ribonuclease Inhibitor (Promega, catalog number: N2111 or N2511) , storage: -20 °C (For probe synthesis)
    13. DTT (Promega, catalog number: P1171), storage: -20 °C (For probe synthesis)
    14. Transcription Optimized 5x Buffer (Promega, catalog number: P1181), storage: -20 °C (For probe synthesis)
    15. T7/SP6/T3 RNA polymerase (Promega, catalogue number, respectively: P2075/ P1085/ P2083), storage: -20 °C (For probe synthesis)
    16. RQ1 RNase-free DNase (Promega, catalog number: M6101), storage: -20 °C (For probe synthesis)
    17. Illustra ProbeQuant G-50 Micro Columns (GE Healthcare, catalog number: 28-9034-08) (For probe synthesis)
    18. 1x PBST (see Recipes)
    19. 8 M Urea stock solution (see Recipes)
    20. 20x SSC (Sodium Saline Citrate or Standard Saline Citrate) (see Recipes, or from Sigma-Aldrich, catalog number: S6639), storage: RT [Controls stringency (annealing of probe to target)]
    21. Wash I (see Recipes)
    22. Wash II (see Recipes)
    23. Wash III (see Recipes)
    24. Urea-based 100% Hybridization Buffer (see Recipes)
    25. Urea-based 50% Hybridization Buffer (see Recipes) 

  6. Antibody incubation
    1. Anti-Digoxigenin-AP, Fab fragments (Roche Applied Sciences, Sigma-Aldrich, catalog number: 11093274910 ), storage: -20 °C [Recognizes the DIG hapten, conjugated to Alkaline Phosphatase (AP)]
    2. Anti-Digoxigenin-POD, Fab fragments (Roche Applied Sciences, Sigma-Aldrich, catalog number: 11207733910 ), storage: -20 °C [Recognizes the DIG hapten, conjugated to horseradish Peroxidase (POD)]
    3. Blocking reagent (Sigma-Aldrich, catalog number: 11096176001), storage: 20-25 °C (To decrease non-specific binging of antibodies)
    4. Maleic acid (Sigma Aldrich, catalog number: M0375), storage: RT (For preparing MAB)
    5. NaCl (Sigma-Aldrich, catalog number: S9888), storage: RT (Salt. For preparing MAB)
    6. NaOH (Sigma-Aldrich, catalog number: 221465), storage: RT (For preparing MAB)
    7. 10x blocking reagent stock solution (see Recipes), storage: -20 °C
    8. 10x MAB (Maleic Acid Buffer) stock solution (see Recipes) (For diluting blocking buffer, used after SSC washes)
    9. 1x MABT buffer (see Recipes)
    10. 10x blocking reagent stock solution (see Recipes), storage: -20 °C
    11. 1x Blocking buffer (see Recipes)

  7. Detection
    1. Aluminum foil (For protecting samples during staining step) 
    2. BCIP (5-Bromo-4-Chloro-3-Indolyl-Phosphate)/NBT (Nitro Blue Tetrazolium) Color Development Substrate (Promega, catalog number: S3771), storage: 4 °C or -20 °C [Substrate of alkaline phosphatase which generates a dark (blue/violet) precipitate]
    3. NaCl (Sigma-Aldrich, catalog number: S9888), storage: RT (Salt. Component of NTMT buffer)
    4. MgCl2 (Sigma-Aldrich, catalog number: M8266), storage: RT (Component of NTMT buffer, MgCl2 is a necessary cofactor for alkaline phosphatase)
    5. Tris-HCl (Tris hydrochloride) (Sigma, catalog number: T3253), storage: RT (pH buffering component of NTMT)
    6. Tween-20 (Sigma-Aldrich, catalog number: P1379), storage: RT
    7. Levamisole (Agilent Dako, catalog number: X302130-2), storage: 2-8 °C (Alkaline Phosphatase inhibitor)
    8. TSA Plus Fluorescence Amplification kit Cyanine 3/5 (Perkin Elmer, Waltham, MA; catalog number: NEL752001KT), storage: 4 °C [Tyramide Signal Amplification (TSA) kit uses Horseradish Peroxidase (HRP or Horseradish POD) to catalyze the deposition of a fluorophore-labeled radicals]
    9. H2O2 (Sigma-Aldrich, catalog number: 516813), storage: 4 °C (Substrate for horseradish peroxidase)
    10. Filters for syringe (Millex-GS, catalog number: SLGS033SS) (For filtering NTMT buffer, in order to avoid precipitates)
    11. Disposable syringes (e.g., 50 ml from Thermo Scientific, catalog number: S7510-50) (For filtering NTMT buffer, in order to avoid precipitates)
    12. 5 M NaCl stock solution (For preparing NTMT buffer, see Recipes), storage: RT
    13. 1 M Tris-HCl, pH 9.5 stock solution (For preparing NTMT buffer, see Recipes), storage: RT
    14. 1 M MgCl2 stock solution (For preparing NTMT buffer, see Recipes), storage: RT
    15. NTMT buffer (see Recipes)
    16. NTMT minus buffer (see Recipes)
    17. NBT/BCIP in NTMT buffer (see Recipes)

  8. Storage & Mounting
    1. Glycerol (Sigma-Aldrich, catalog number: G5516), storage: RT (Mounting medium)
    2. Citifluor-AF1 (Citifluor, catalog number: AF1-100), storage: RT [Mounting medium for fluorescent samples (on-hardening antifading/anti-bleaching)]
    3. Slide (e.g., 76 x 26 mm, with frosted end (practical for labeling), from Knittel Glass (Mounting)
    4. Coverslide (e.g., 20 x 20 mm, thickness 1, from Knittel Glass) (Mounting)
    5. Nailpolish (For sealing mounted specimens; any commercial nailpolish can be used, however prefer a transparent one, and test first on lesser important samples, in case it contains too much solvent–which might damage samples)

Equipment

Note: No specific brand/model is provided; this equipment is commonly available in laboratories.

  1. Pipettes 
  2. Heating block (For probe denaturation, e.g., featuring a temperature range 10-100 °C)
  3. Metallic forceps (For transferring baskets, e.g., from Fine Science Tools)
  4. Waterbath/Hybridization oven (Hybridization and stringent washes)
  5. Glass bottles with high-temperature resistant screw caps (e.g., 250 ml bottle from Fisherbrand (catalogue number: FB-800-250); 500 ml bottle from VWR [borosilicate 3.3; catalogue number: 215-1594); 1000 ml bottle from Scott Duran (Original GL 45 series)]
  6. pH meter (For adjusting pH of solutions)

Procedure

General notes on samples handling:

  1. Sample fixation is best performed in tightly sealed RNase free containers, such as Eppendorf or Falcon tubes. 
  2. Once samples are re-hydrated, they can be transferred to RNase free in situ hybridization baskets (shown in Figure 1A).
  3. The density of samples might affect the in situ hybridization results: overfilled vials/baskets often yield poorly stained/damaged samples, and too little amount of sample (e.g., one embryo) might become over-stained (possibly due to unbalanced ratio probe/target).

  1. Sample fixation/dehydration (Table 1)

    Notes:

    1. Fixatives should be handled and disposed of according to laboratory safety guidelines.
    2. Fixation (choice of fixative, duration of fixation) must be adapted to sample types. 
    3. Two alternative types of fixation are provided, for colorimetric in situ hybridization (CISH), and for fluorescent in situ (FISH).

      Table 1. Fixationa


  2. Rehydration (Table 2)

    Table 2. Rehydration


  3. (Optional) Permeabilization; proteinase K treatment permeabilize tissues (Table 3)
    Notes:
    1. Treatment time should be titrated for each new batch of enzyme, as strength might vary. A useful rule of thumb is treating a test sample until tissue damage appears, and halving the resulting time (see Figure 3 in Notes section for further details). 
    2. This step might prove unnecessary for very delicate tissues.

    Table 3. Permeabilization


  4. (Optional) Acetylation (Table 4)
    Note: This step might help reducing non-specific signal, due to probes binding to positively charged surfaces.

    Table 4. Acetylation


  5. (Optional) Re-fixation (Table 5)
    Notes:
    1. Depending on sample type (e.g., fragile invertebrate embryos), an additional re-fixation step prior to hybridization might be required. 
    2. Generally, it is advisable to re-fix after proteinase K treatment. 
    3. Glutaraldehyde might be omitted: it is recommended to test several options while setting up the protocol for the first time. Glutaraldehyde penetrates tissues slowly, and its use is preferable for samples thinner than 1 mm (Eltoum et al., 2001). 

    Table 5. Re-fixation


  6. Hybridization (Table 6)
    Notes:
    1. Do not let the temperature drop during the pre-hybridization/hybridization phase. Transfer of baskets or addition of probe might take long time. When working on multiple samples, set up manageable batches.
    2. The urea-based hybridization buffer tends to be viscous, and evaporation can be an important issue, leading to the formation of crystals that will damage the samples. Seal vials/multi-well plate carefully (Figure 1C).
    3. Be aware that some sample types might become temporarily transparent while in heated hybridization buffer. In this phase, they will be also more fragile.

    Table 6. Hybridization



  7. Blocking (Table 7)

    Table 7. Blocking


  8. Signal detection
    AP antibody, colorimetric reaction (Table 8)

    Table 8. Colorimetric signal detection


    Fluorescent detection (POD antibody, signal amplification) (Table 9)

    Table 9. Fluorescent signal detection


  9. CISH mounting (Table 10)

    Table 10. Colorimetric samples- mounting


  10. FISH mounting (Table 11)

    Table 11. Fluorescent samples- mounting

Data analysis

The urea-variant in situ hybridization protocol generates reliable results. Sample morphology is well preserved, and the good ratio signal-to-background allows for detecting of gene expression with cellular resolution. Figure 2 depicts the results of colorimetric (CISH) and fluorescent (FISH) protocols performed on a Clytia medusa.
  Please refer to the original paper (Sinigaglia et al., 2018) for a further comparison of the formamide-based and urea protocol, for examples from diverse species, and for a more detailed analysis of results.


Figure 2. Detection of gene expression in medusae. RFamide (accession number for Clytia gene: KX496951) is expressed in nervous system cells. The urea- in situ hybridization protocol detects cells RFamide+, scattered thorough the medusa body; here a detail is shown, including (left to right): tentacle, tentacle bulb, circular canal running at the periphery of the umbrella, and umbrella. Tissues are well preserved, in particular the fragile umbrella. A. CISH result, the focus is on the scattered cells in the tentacle and in the circular canal. Image taken with a Zeiss Axio Imager A2. B. FISH results, maximum projection (done with Fiji) of a z-stack image taken with a Leica SP8 confocal microscope. Both images were cropped; scale bars = 50 μm.

Notes

  1. Controls
    1. It is highly recommended to perform both a negative control (e.g., using sense probes, or a probe of similar length for a gene not expressed in the sample being studied), and a positive control (using a reliable, known antisense probe), in particular when testing any new probe or optional step.
    2. When testing for the first time this urea-based protocol, it is recommended to perform a parallel in situ hybridization with an eventual previous formamide-based protocol (if one is already in use in the laboratory).

  2. mRNA probes
    1. Riboprobes (mRNA probes) can be synthesized from either a linearized template plasmid, or from a PCR product. The DIG-labeled RNA probes shown in the original research paper (Sinigaglia et al., 2018), including the RFamide probe employed in the present protocol, were synthesized using one of the RNA polymerase kits from Promega (T7, SP6 or T3 polymerases, according to the orientation of insert). For probe synthesis, add in order: 2 μl of 10x DIG labeling mix RNA (Roche); 0.5 μl of RNasin Ribonuclease Inhibitor (Promega) ; 2 μl of 100 mM DTT (Promega); 4 μl of Transcription Optimized 5x Buffer (Promega) ; 1-2 μg of DNA template (calculate the appropriate volume); RNase-free H2O to a 20 μl of total volume ; 0.5 μl of T7/SP6/T3 RNA polymerase (Promega). Run reaction for 2-5 h at 37 °C, then arrest by digesting the DNA template with 1.5 μl of RQ1 RNase-free DNase (Promega), at 37 °C for 30 min. Elute by adding 30 μl of RNase free H2O, purify with the illustra ProbeQuant G-50 Micro Columns (GE Healthcare). Check probe integrity by running 1 μl of purified probe on 1% agarose gel, and quantify with nanodrop. Probes can be stored at -20 °C or -80 °C, addition of 50% (v/v) RNase-free formamide helps preserving RNA integrity.
    2. It is recommended to synthesize always both the antisense (complementary to mRNA) probe and the sense probe, to be used as a control for the specificity of binding. 
    3. Probes are usually designed on the coding sequence of a gene, but they can also target the 3' UTR region (this might be helpful for example when discriminating highly similar genes). Clone the desired region from cDNA, possibly deriving from mRNA extracted from the stage/body region of interest (different stages/tissues might express differently-spliced forms of the same gene).
    4. Recommended probe length ranges between 500 and 800, up to 1,500 bp. Long probes can be fragmented, e.g., by alkaline hydrolysis.
      Alkaline hydrolysis (Cox et al., 1984): Hydrolyze probe by adding 1:1 volume of hydrolysis buffer (40 mM NaHCO3/60 mM Na2CO3, pH 10.2) freshly prepared in RNase-free water. Incubate at 60 °C, for a length of time (expressed in minutes) calculated as:


      Where, L0 is original transcript length (expressed in kb) and Lf is the desired fragment length (in kb).
      Neutralize by adding sodium acetate (to 0.1 M final, at pH 6.0), and glacial acetic acid (to 0.5%, v/v), and precipitate with ethanol.

  3. Adapting the protocol/Variants
    1. This protocol is based on Clytia in situ hybridization (Sinigaglia et al., 2018); it is however meant to represent a general guideline. When adapting it to new study organisms/tissues/cells, it is recommended to start by simply substituting the formamide in the original hybridization buffer, with a urea solution, to a final concentration of 4 M.
    2. Variants to the protocol are possible, in particular the fluorescent in situ hybridization has already been successfully coupled to immunostaining or to EdU reactions (data not shown).
    3. This protocol might be used also on tissue sections (frozen or paraffin-embedded), in which case further adjustments might be necessary (e.g., reducing detergents or urea concentration in the hybridization buffer).
    4. Urea-based hybridization buffer might also be tested with smFISH.
    5. Proteinase K treatment is optional. Proteinase K digests proteins, thus permeabilizing tissues, and aiding the penetration of probes and reagents. Too strong treatment might damage tissue morphology, while insufficient digestion might result in poor detection of signal. Both incubation time and enzyme concentration should be assessed for each new batch of enzyme.


      Figure 3. Titration of proteinase K treatment. Images show the progressive digestion of a fixed Clytia medusa, treated with 10 μg/ml of proteinase K in 1x PBST. Visible damages (yellow asterisk) start to appear after 30 min of treatment, and by 40 min the medusa is severely damaged (notice the detached velum and gonad). According to the guideline provided in the main protocol (see Table 3), the proteinase K treatment should last for 15-20 min during in situ hybridization. 

  4. Troubleshooting
    1. Damaged samples
      1. Make sure that samples are always covered by solution, and never let them dry. 
      2. Verify that hybridization buffer did not evaporate.
      3. Add a second fixation step prior to hybridization, after rehydration.
      4. Remove permeabilization step, or eventually reduce duration of treatment.
      5. Handle samples and baskets carefully. Samples might be more fragile while in methanol and in hybridization solution.
      6. If damages are due to crystals forming on samples, try rapidly washing samples with MilliQ H2O.
    2. Absent/poor signal
      1. Verify probe quality (check for degradation) and that correct antisense probe is being used. Long probes usually perform worse, the optimal length is however species- and gene-specific (aim for 500-800 bp). Probes can be fragmented (e.g., by alkaline hydrolysis, see point B4 of Notes section); this will improve both penetration and recognition of target. 
      2. Add permeabilization step/re-test/renew proteinase K batch.
      3. Increase/reduce probe concentration in hybridization mix.
      4. In case of CISH: extend the signal detection step (renew frequently the NBT/BCIP mix, while keeping samples at 4 °C). In case of FISH: extend the signal detection reaction, or repeat it.
      5. In case of CISH: use freshly prepared staining solution. Freshly prepare also NTMT (do not use if older than a day, keep at 4 °C while not in use). 
      6. In case of FISH: prepare buffer just before use.
      7. Increase/reduce urea concentration in the hybridization mix (8 M to 2 M): in general, higher concentrations will have a stronger permeabilizing effect, and are recommended for difficult/hard tissues.
      8. Verify the pH of solutions, in particular of TEA and of SSC stock solutions.
      9. Samples might be over-fixed, verify. It might be advisable to remove glutaraldehyde, in case it was added to the fixative solution.

    3. Non-specific signal
      1. In case of CISH: add levamisole to NTMT and staining mix.
      2. In case of CISH: extend incubation time with NTMT, prior to NBT/BCIP color reaction.
      3. In case of CISH: protect NBT/BCIP mix from light. Verify that the stock solution did not turn purple or black and that it appears homogeneous. 
      4. In case of CISH: try performing FISH instead (allows for more precise observation, avoids eventual issues with non-specific deposition of precipitate).
      5. Pre-hybridize overnight.
      6. Add acetylation step.
      7. Make sure that acetic anhydride solution (Steps 1 and 2 in Procedure D) gets efficiently mixed, and that samples get well in contact with it.
      8. Increase hybridization temperature.
      9. Reduce probe concentration. Recycle probe (store the Hybridization mix at -20 °C).
      10. Increase duration/stringency of post-hybridization washes. Stringency is modulated by changing the concentration of SSC buffer (5-6x for low stringency, 2-0.1x for high stringency).
      11. Make sure that no dust/dirt is attached to specimens before starting the color reaction: particles might derive from insufficient washing of recycled baskets, from solutions (filter them, in particular NTMT solution), from clothes. If present, remove them manually.
        In general, particles attached to specimens most commonly derive from old solutions: renew them, in particular the staining reagents, and adjust pH accurately. A step in NTMT minus buffer (filtered) prior to staining might also help
      12. In case of CISH: extend Steps 9 in Procedure H (Colorimetric detection) and Procedure I (CISH mounting), keeping samples at 4 °C.
      13. Verify the pH of solutions, in particular of TEA and of SSC stock solutions.

    4. Signal too strong/rapid
      1. Reduce probe concentration.
      2. For CISH: dilute staining solution. Reduce staining time. Add levamisole to staining solution and/or carry out staining reaction at 4 °C, in order to slow down AP reaction.
      3. In case of CISH: try performing FISH instead (allows for more precise observation, avoids eventual issues with non-specific deposition of precipitate).
      4. Increase the stringency of washes.
      5. Add acetylation step.

Recipes

  1. 10x PBS (pH 7.4) stock solution
    1. Weigh and add: 25.6 g of Na2HPO4·7H2O, 80 g of NaCl, 2 g of KCl, 2 g of KH2PO4
    2. Bring to 1 L with MilliQ H2O
    3. Autoclave
    4. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  2. 1x PBST
    1. Add 0.1% Tween-20 to 1x PBS solution
    2. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated) 
  3. HEM buffer
    0.1 M HEPES, pH 6.9
    50 mM EGTA, pH 7.2
    10 mM MgSO4
    Prepare freshly
  4. CISH fixation buffer
    3.7% formaldehyde plus
    0.4% glutaraldehyde
    1x PBS (pH 7.4)
    Prepare freshly
  5. FISH fixation buffer
    3.7% formaldehyde in HEM buffer
    Prepare freshly
  6. Dehydration buffer
    50% 1x PBST
    50% Methanol
    Prepare freshly
  7. Rehydration buffer
    50% Methanol
    50% 1x PBST
    Prepare freshly
  8. Permeabilization buffer
    10 μg/ml proteinase K in 1x PBST
    Prepare freshly
  9. Neutralization buffer
    2 mg/ml glycine in 1x PBST
    Prepare freshly
  10. 10x TEA stock solution
    1. Prepare 1 M solution in H2O
    2. Adjust the pH to 7.8
    3. Do not autoclave
    4. Handle TEA with care (do not shake)
    5. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated) 
  11. 0.1 M TEA
    Dilute 10x TEA stock solution with 1x PBST, to 0.1 M Triethanolamine (TEA)
    Prepare freshly
  12. 0.25% Acetic anhydride in 0.1 M TEA
    Add 0.25% acetic anhydride (v/v) to 0.1 M TEA solution, and mix thoroughly
    Prepare freshly
  13. Re-fixation buffer
    3.7% formaldehyde plus 0.2% glutaraldehyde in 1x PBST
    Prepare freshly
  14. 8 M Urea stock solution
    1. Dissolve urea in MilliQ H2O, calculating the required amounts (MW = 60.06 g/mol)
    2. Be aware that the reaction is endothermic
    3. Prepare freshly and sterilize by filtration if necessary
    4. The solution can be used for both hybridization and post-hybridization steps, in this case keep at 4 °C
  15. 20x SSC (sodium saline citrate) stock buffer (1 L)
    Note: 20x concentration of buffer prevents fungal and bacterial growth.
    1. Dissolve 175.3 g NaCl and 88.2 g of trisodium citrate (Na3C6H5O7) in 900 ml MilliQ H2O
    2. Adjust the pH to 7.0, top to 1 L with MilliQ H2O
    3. Autoclave to sterilize
    4. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  16. 20% SDS (Sodium Dodecyl Sulfate)
    Precipitation can occur at low temperatures. In this case, just let the buffer warm up before use
  17. Urea-based Hybridization Buffer
    Final concentration:
    5x SSC
    1% dextran
    4 M urea
    50 μg/ml of heparin
    50 μg/ml of tRNA (or salmon sperm)
    1% SDS
    To prepare (10 ml) add in the following order, prepare freshly:
    2.5 ml of 20x SSC
    0.1 g of dextran powder (dissolve well)
    5 ml of 8 M urea stock solution
    25 μl of heparin (20 mg/ml stock)
    10 μl tRNA
    5.1 ml of MilliQ H2O
    500 μl of 20% SDS
    Notes:
    1. Be aware that premature addition of SDS might cause a precipitate to appear.
    2. Do not agitate vigorously, to avoid bubbles.
    3. Hybridization buffer can also be aliquoted and stored at -20 °C, in this case avoid multiple thawing/freezing cycles.
  18. 50% Hybridization Buffer
    50% Urea-based Hybridization Buffer
    50% 1x PBST
    Prepare freshly
  19. Wash I
    Final concentration:
    4 M urea
    0.1% Tween-20
    5x SSC
    Add MilliQ H2O to volume
    Prepare freshly; or shortly beforehand (e.g., the evening before use)
  20. Wash II
    Final concentration:
    2 M urea
    0.1% Tween-20
    2x SSC
    Add MilliQ H2O to volume
    Prepare freshly; or shortly beforehand (e.g., the evening before use)
  21. Wash III
    Final concentration:
    0.1% Tween-20
    2x SSC
    Add MilliQ H2O to volume
    Prepare freshly; or shortly beforehand (e.g., the evening before use)
  22. 10x MAB buffer stock solution
    1. Add to 700 ml of MilliQ H2O: 116 g of maleic acid, 87 g of NaCl, 40 g of NaOH
    2. Adjust carefully the pH to 7.5, and top to 1 L with MilliQ H2O
    3. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  23. 1x MABT buffer
    1. Dilute stock solution 1:10 with MilliQ H2O, add 0.1% Tween-20
    2. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  24. 10x blocking reagent stock solution
    1. Dissolve in maleic acid buffer to a final concentration of 10% (w/v), shaking and heating to 60 °C
    2. Stock solution should be autoclaved, aliquoted and stored at -20 °C
  25. 1x Blocking buffer
  26. 5 M NaCl
    1. Dissolve 292 g of NaCl in 800 ml of MilliQ H2O
    2. Adjust the volume to 1 L with MilliQ H2O
    3. Autoclave for sterilizing
    4. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  27. 1 M Tris-HCl, pH 9.5
    1. Dissolve 121.1 g of Tris-base in 800 ml of MilliQ H2O
    2. Adjust the pH to 9.5 by adding HCl (let the solution reach room temperature for accurate adjustment of pH)
    3. Adjust the volume to 1 L with MilliQ H2O
    4. Autoclave for sterilizing
    5. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  28. 1 M MgCl2
    1. Dissolve 203.3 g of MgCl2·6H2O in 800 ml of MilliQ H2O
    2. Adjust the volume to 1 L with MilliQ H2O
    3. Autoclave
    4. Store at room temperature, discard if precipitate/particles appear (the bottle might have been contaminated)
  29. NTMT buffer
    Final concentration:
    100 mM NaCl
    100 mM Tris-HCl
    50 mM of MgCl2
    For 100 ml, add:
    2 ml of 5 M NaCl
    10 ml of 1 M Tris-HCl (pH 9.5)
    5 ml of 1 M MgCl2
    1 ml of Tween-20
    MilliQ H2O to volume
    Prepare freshly
  30. NTMT minus buffer
    Final concentration:
    100 mM NaCl
    100 mM Tris-HCl
    For 100 ml add:
    2 ml of 5 M NaCl
    10 ml of 1 M Tris-HCl (pH 9.5)
    1 ml of Tween-20
    MilliQ H2O to volume
    Prepare freshly
  31. NBT/BCIP in NTMT buffer
    1. Add to NTMT buffer:
      0.08 mg/ml of Nitro Blue Tetrazolium
      0.1 mg/ml of 5-Bromo-4-Chloro-3-Indolyl-Phosphate
    2. Prepare freshly and protect from light

Acknowledgments

I thank Lucas Leclère and Evelyn Houliston for comments on the manuscript and discussions, and the other members of the Houliston/Momose group, in particular Sandra Chevalier, for discussion and assistance. I also thank all the people who contributed to testing this protocol on their organism of study, in particular Gonzalo Quiroga-Artigas, Lorenzo Ricci, Daniel Thiel. I thank the anonymous reviewers for their helpful comments on the manuscript. I also thank Enrique Arboleda for comments on the final version of the manuscript. Funding: Agence National de la Recherche, grant “MEDUSEVO” ANR-13-PDOC-0016.

Competing interests

The author declares there are no competing interests.

References

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  16. Lambert, D. and Draper, D. E. (2012). Denaturation of RNA secondary and tertiary structure by urea: simple unfolded state models and free energy parameters account for measured m-values. Biochemistry 51(44): 9014-9026.
  17. Leclère, L., Horin, C., Chevalier, S., Lapébie, P., Dru, P., Peron, S., Jager, M., Condamine, T., Pottin, K., Romano, S., Steger, J., Sinigaglia, C., Barreau, C., Quiroga Artigas, G., Ruggiero, A., Fourrage, C., Kraus, J. E. M., Poulain, J., Aury, J. M., Wincker, P., Queinnec, E., Technau, U., Manuel, M., Momose, T., Houliston, E. and Copley, R. R. (2019). The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle. Nat Ecol Evol 3(5):801-810.
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简介

原位杂交方法通常被用于检测核酸序列,从而定位基因表达或研究染色体的组织结构。这些方法依赖于标记探针与目标内源性核酸序列(杂交步骤)的成对结合,然后通过荧光或比色反应检测退火序列。成功的杂交需要组织的渗透性,然后是核酸链的变性,通常在甲酰胺基缓冲液中和高温下进行。这种反应条件,除了对健康造成危害(包括操作和废物处理),对某些物种或发育阶段的脆弱组织来说,可能过于苛刻。我们在这里详细介绍了原位杂交的另一种方法,即用尿素溶液代替有毒的甲酰胺。这种替代物改善了几种动物的组织保存和信噪比检测。本文描述的方案最初是为水母clytia hemisphaerica开发的,它提供了将基于甲酰胺的传统方案应用于尿素变体的指南。基于尿素的协议已经成功地应用于各种无脊椎动物和脊椎动物物种,显示了这种修改的简便性,并为科学界提供了一个有前途的、安全的和多用途的工具。
【背景】单链核酸序列的成对互补结合激发了一种强大的原位杂交技术的发展,这种技术允许研究人员在细胞和组织环境中可视化DNA或RNA链的位置(Pardue and Gall,1969)。此后,在外源互补探针的标记(例如>,放射性或半抗原为基础)、样品类型(例如>,整个胚胎或组织切片)和检测方法(例如>,荧光或染色)方面,已经发展出许多不同的变体。测量信号)。



探针的精确退火取决于目标序列的可接近性,通过组织和细胞膜的适当渗透性(允许试剂渗透)和核酸链的变性(暴露互补的目标序列)来实现。要获得足够的检测灵敏度、退火的特异性和样品形态的保存,往往需要对反应条件进行耗时的优化。核酸链的有效变性和退火通常是通过高温和在杂交缓冲液中加入变性剂来实现的。理想情况下,反应温度应与感兴趣序列的碱基组成相适应:杂交温度比变性温度低25°C左右(TM;Marmur和Doty,1961)。50-70%的甲酰胺通常用作变性试剂,这一选择自80年代以来基本上没有受到质疑,只有少数例外,例如使用10-15%甲酰胺的SMFISH(单分子荧光原位杂交法)(Haimovich和Gerst,2018)。



高温增加了杂交缓冲液的蒸发,使甲酰胺(一种刺激性、胚胎毒性和致畸性溶剂)已经造成的健康风险更加复杂(Gleich,1974;Stula和Krauss,1977;Merkle和Zeller,1980;Kennedy和Short,1986;Fail等人,1998;George等人,2000年和2002年;另见Sinigalia等人,2018年)。杂交样品和废物需要适当处理(cicad 31),从而限制了标准甲酰胺协议在敏感环境中的应用,如怀孕或在教室中。



在早期,人们发现多种溶剂可以有效地破坏核酸链的稳定性,而尿素的稳定性尤为明显(Herskovits,1963)。事实上,尿素和甲酰胺具有相似的性质,并且在许多应用中被等效地使用,例如细菌荧光原位杂交(Fontenet等,2016年)、蛋白质变性和成像用清除剂(在Azaripour等中进行了综述>2016年)。尿素可以有效地替代甲酰胺在北方和南方的印迹实验,特别是在浓度为2 m-4 m的范围内(simard等,2001)。尿素的作用机理还相对不清楚。尿素降低每摩尔尿素约2°C的DNA的Tm(Hutton,1977),因此比每摩尔尿素降低2.4-2.9°C的甲酰胺效率稍低。尿素分子与RNA的结合较弱,破坏其碱基对相互作用,最终破坏RNA分子的结构(Herskovits和Bowen,1974;Priyakumar等人,2009;Lambert和Draper,2012)。



以粉末或晶体形式存在的尿素是水溶性的,在20℃下可以容易地制备8 m储备溶液。溶液往往是粘性的(Hutton,1977),这可能解释了为什么高浓度尿素的效果较差(Simard等人,2001)。这种粘度的增加需要在原位杂交过程中小心,以确保样品被有效浸泡,蒸发量最小化。



在不同的后生动物物种和不同的发育阶段,本文提出的基于尿素的方案比基于标准甲酰胺的方案更为有效(见Sinigalia等人,2018年)。检测灵敏度得到提高,使不可置信的复杂基因表达模式可视化(Sinigglia等,2018年)。这种检测灵敏度的增加可以解释为尿素对组织的额外渗透作用(Limet al>,2009;Huanget al>,2011),可能是由于细胞内渗透压的增加(在Tainakaet al>,2016年进行了综述)。原始研究论文(Sinigaliaet al>,2018)显示,组织的过度水化也可能部分解释了精细样品形态的改善。



这里详细说明的方案最初是为水螅水母clytia hemisphaerica>(quiroga artigaset al>,2018年;sinigaliaet al>,2018年,lecl_reet al>,2019年制定的)。根据这些指南,已经为后生动物物种和发育阶段的多样性制定了尿素替代方案,包括原生动物和后生动物胚胎(Thielet a l>,2017;Sinigaliaet al>,2018)、AcoelHofstenia miamia>(L.Ricci,个人交流),水母aurelia aurita(m.manuel和t.condamine,个人交流),和桨鱼(m.minarik,个人交流)。用尿素替代甲酰胺也应用于小鼠卵母细胞上的DNA-FISH(Manil Segalen等人,2018年),进一步显示了这种方法的多功能性。发现多功能性源于关键实施的简单性,即用等量尿素溶液(最终浓度为4 m)替代甲酰胺。当将先前的方案适应于新的物种或组织时,建议从简单地将甲酰胺转换为尿素开始,剩下的步骤保持不变。还提供了最终故障排除指南,为成功的原位杂交提供了一般建议。

关键字:RNA原位杂交, 尿素, 甲酰胺, 健康和安全, 信噪比

材料和试剂

  1. 多步骤使用的材料和试剂
    1. 手套
    2. 无核糖核酸酶试管(例如>,1.5毫升Eppendorf试管,或等效物)
    3. 原位篮(样品容器,如图1a所示)
      它们可以是netwell篮(康宁),也可以是由尼龙网和微型离心管构成的自制篮,如sive等人所述。(2007年)。篮的大小取决于样品的类型;它们通常适合6、12或24孔板的井内。建议在篮(而不是试管)中进行原位杂交:
      1. 减少因移液管而造成的样品损失和损坏。
      2. 为了加速溶液的交换(篮子可以在镊子的帮助下简单地转移到带有新溶液的多孔板上(图1b)–在这种情况下,轻轻敲击篮子,使其紧靠井壁,以确保清除旧溶液,然后轻轻地将其浸入新井中)。
      3. 篮子可以循环多次,在彻底清洗后(e.g.>,用milliq h2o冲洗,然后浸入1 M NaOH溶液中和RNase,并用无RNase的milliq h2o冲洗几次-只需将它们放在烧杯中的摇床上即可确保水平旋转)。


        图1。样品载体。a.三个网底篮子的示例。b.24孔板,用于原位杂交。c.相同的板,封闭并用塑料薄膜包裹,以避免杂交步骤中的蒸发。

    4. 多孔板(用于安装原位篮,如图1b所示);(例如,24孔细胞培养板,无菌,带盖,来自Cellstar(格雷纳生物一号,目录号:662-160)
    5. 冰和冰容器
    6. 毫希2o(储存:室温)
    7. 吐温-20(Sigma-Aldrich,目录号:p1379),储存:RT(温和洗涤剂)。鉴于其高粘度,建议制备10%的工作溶液)
    8. Na2HPO4·7h2O(Sigma Aldrich,目录号:431478),储存:RT(用于制备10x PBS溶液)
    9. NaCl(Sigma-Aldrich,目录号:S9888),储存:RT(用于制备10X PBS溶液)
    10. KCL(Sigma-Aldrich,目录号:P9333),储存:RT(用于制备10倍PBS溶液)
    11. kh2po4(sigma-aldrich,目录号:p9791),存储:rt(用于制备10x pbs溶液)
    12. 10x PBS(磷酸盐缓冲盐水)pH值7.4(见配方,或Calbiochem药片,目录号:524650),存储:RT(有助于保持恒定pH值)

  2. 固定
    1. 37%甲醛(Sigma-Aldrich,目录号:252549),储存:RT(交联)
    2. 8%戊二醛(电子显微镜科学,目录号:16020),储存温度:4℃(交联)
    3. egta(乙二醇双(2-氨基乙醚)-n,n,n',n'-四乙酸)(sigma-aldrich,目录号:e3889),储存:rt[钙/镁] 螯合剂,可降低酶活性(如rnase);hem缓冲液试剂]
    4. HEPES(Sigma-Aldrich,目录号:H4034),贮存:RT(HEM缓冲液试剂)
    5. mgso4(sigma-aldrich,目录号:m7506),存储:rt(hem缓冲液试剂)
    6. 吐温-20(Sigma-Aldrich,目录号:p1379),存储:RT
    7. 甲醇(例如>,Sigma-Aldrich,目录号:322415),储存:RT
    8. 1x PBS(见配方)
    9. 1x PBST(见配方)
    10. CISH固定缓冲液(见配方)
    11. 鱼固定缓冲液(见配方)
    12. 脱水缓冲液(见配方)
    13. 补液缓冲液(见配方)
    14. 再固定缓冲液(见配方)

  3. 乙酰化
    1. tea(三乙胺)(sigma-aldrich,目录号:t0886),存储:rt(用于减少带负电探针与组织的非特异性结合)
    2. 醋酸酐(Sigma-Aldrich,目录号:A6404),储存温度:4℃(用于减少带负电探针与组织的非特异性结合)
    3. 1x PBST(见配方)
    4. 10倍茶原液(见配方)
    5. 0.1米茶(见食谱)
    6. 0.1米茶叶中0.25%醋酸酐(见配方)

  4. 渗透性
    1. 蛋白酶K(Sigma-Aldrich,目录号:P4850),储存温度:4°C或-20°C(消化蛋白质,包括核酸酶)。最适ph值为7.5-9.0)
    2. 甘氨酸(Sigma-Aldrich,目录号:G8898),储存:RT(用于中和蛋白酶K)
    3. 1x PBST(见配方)

  5. 杂交
    1. 塑料薄膜(用于杂交步骤中包装样品容器)
    2. 尿素(Sigma-Aldrich,目录号:U5378),储存:RT(变性剂)
    3. 右旋糖酐硫酸钠,mr~200'-00(sigma-aldrich,目录号:67578-25g),存储:rt[增加分子拥挤,因此可以加速杂交动力学(局部增加探针浓度)]
    4. trna,来自面包酵母(sigma-aldrich,目录号:10109509001),储存温度:-20°C(阻止非特定探针结合)
    5. 鲑鱼精子(Thermo Fisher,目录号:156312011),储存温度:-20℃(阻止非特异性探针结合)
    6. 肝素钠(Thermo Fisher,目录号:10239840),储存温度:-20°C(通过与与DNA结合的蛋白质结合,有助于降低背景温度)
    7. 20%十二烷基硫酸钠(Fisher,目录号:BP131),储存:RT(洗涤剂)。渗透膜,促进探针分布)
    8. NaCl(Sigma-Aldrich,目录号:S9888),储存:RT(盐。准备20X SSC储备缓冲液,见配方)
    9. 柠檬酸三钠(Na3C6H5O7)(制备20X SSC储备缓冲液,见配方)
    10. 吐温-20(Sigma-Aldrich,目录号:p1379),存储:RT
    11. 10x dig标记混合rna(罗氏,目录号:11277073910),储存温度:-20℃(用于探针合成)
    12. rnasin核糖核酸酶抑制剂(promega,目录号:n2111或n2511),储存温度:-20°C(用于探针合成)
    13. DTT(Promega,目录号:p1171),存储温度:-20°C(用于探针合成)
    14. 转录优化5x缓冲液(Promega,目录号:p1181),存储温度:-20℃(用于探针合成)
    15. T7/SP6/T3 RNA聚合酶(Promega,目录号分别为:p2075/p1085/p2083),储存温度:-20℃(用于探针合成)
    16. RQ1无核糖核酸酶DNA酶(Promega,目录号:M6101),储存温度:-20°C(用于探针合成)
    17. Illustra Probequant G-50微柱(GE Healthcare,目录号:28-9034-08)(用于探针合成)
    18. 1x PBST(见配方)
    19. 8 m尿素储备溶液(见配方)
    20. 20X SSC(枸橼酸钠或标准枸橼酸盐)(见配方,或来自Sigma-Aldrich,目录号:S6639),存储:RT[控制严格性(探头到目标的退火)]
    21. 洗一次(见食谱)
    22. 洗二(见食谱)
    23. 洗三(见食谱)
    24. 尿素基100%杂交缓冲液(见配方)
    25. 尿素基50%杂交缓冲液(见配方)

  6. 抗体孵育
    1. 抗地高辛AP,Fab片段(Roche应用科学,Sigma-Aldrich,目录号:11093274910),储存温度:-20℃[识别地高辛半抗原,与碱性磷酸酶(AP)结合]
    2. 抗地高辛吊舱,Fab片段(罗氏应用科学公司,Sigma-Aldrich,目录号:11207733910),储存温度:-20℃[识别地高辛半抗原,结合辣根过氧化物酶(POD)]
    3. 阻断剂(Sigma-Aldrich,目录号:11096176001),储存温度:20-25°C(减少抗体的非特异性结合)
    4. 马来酸(Sigma-Aldrich,目录号:M0375),储存:RT(用于制备单克隆抗体)
    5. NaCl(Sigma-Aldrich,目录号:S9888),储存:RT(盐。用于制备单克隆抗体)
    6. NaOH(Sigma-Aldrich,目录号:221465),贮存:RT(用于制备单克隆抗体)
    7. 10x封闭试剂储备溶液(见配方),储存温度:-20°C
    8. 10x MAB(马来酸缓冲液)储备溶液(见配方)(用于稀释封闭缓冲液,在SSC清洗后使用)
    9. 1x mAbt缓冲区(见配方)
    10. 10x封闭试剂储备溶液(见配方),储存温度:-20°C
    11. 1x块缓冲区(见配方)

  7. 检测
    1. 铝箔(用于染色步骤中保护样品)
    2. BCIP(5-溴-4-氯-3-吲哚基磷酸酯)/NBT(硝基蓝四氮唑)显色底物(Promega,目录号:S3771),储存温度:4°C或-20°C[产生暗(蓝/紫)沉淀的碱性磷酸酶底物]
    3. NaCl(Sigma-Aldrich,目录号:S9888),储存:RT(盐。ntmt缓冲组件)
    4. mgcl2(sigma-aldrich,目录号:m8266),存储:rt(ntmt缓冲液成分,mgcl2是碱性磷酸酶的必要辅因子)
    5. tris hcl(tris盐酸盐)(sigma,目录号:t3253),储存:rt(ntmt的ph缓冲成分)
    6. 吐温-20(Sigma-Aldrich,目录号:p1379),存储:RT
    7. 左旋咪唑(安捷伦达科,目录号:x302130-2),储存温度:2-8℃(碱性磷酸酶抑制剂)
    8. TSA PLUS荧光放大试剂盒,菁3/5(Perkin Elmer,Waltham,MA;目录号:NEL752001KT),储存温度:4℃【酪酰胺信号放大(TSA)试剂盒使用辣根过氧化物酶(HRP或辣根过氧化物酶)催化荧光标记自由基的沉积】
    9. H2O2(Sigma-Aldrich,目录号:516813),储存温度:4℃(辣根过氧化物酶底物)
    10. 注射器过滤器(Millex GS,目录号:SLGS033SS)(用于过滤NTMT缓冲液,以避免沉淀)
    11. 一次性注射器(例如,>,50毫升,来自Thermo Scientific,目录号:S7510-50)(用于过滤NTMT缓冲液,以避免沉淀)
    12. 5 M NaCl储备溶液(用于制备NTMT缓冲液,见配方),储存:RT
    13. 1 M Tris HCl,pH 9.5储备溶液(用于制备NTMT缓冲液,见配方),储存:RT
    14. 1 m mgcl2储备溶液(用于制备ntmt缓冲液,见配方),储存:rt
    15. ntmt缓冲区(见配方)
    16. ntmt减去缓冲区(见配方)
    17. ntmt缓冲区中的nbt/bcip(见配方)

  8. 存储和安装
    1. 甘油(Sigma-Aldrich,目录号:G5516),储存:RT(安装介质)
    2. citifluor-af1(citifluor,目录号:af1-100),存储:rt[荧光样品的安装介质(硬化、抗老化/抗漂白)]
    3. 从针状玻璃(安装)上滑动(例如,76 x 26 mm,带磨砂端(标签实用)
    4. 盖玻片(例如,>,20 x 20 mm,厚度1,来自针织玻璃)(安装)
    5. Nailpolish(用于密封安装的标本;任何商业指甲油都可以使用,但是更喜欢透明的,并在较小的重要的测试。样品,以防含有过多的溶剂——这可能会损坏样品)

设备

注:不提供特定品牌/型号;实验室通常提供此设备。>

  1. 移液管
  2. 加热块(用于探头变性,例如>,温度范围为10-100°C)
  3. 金属镊子(用于转移篮子,例如>,来自精细科学工具)
  4. 水浴/杂交炉(杂交和严格洗涤)
  5. 带耐高温螺旋盖的玻璃瓶(例如,>,Fisherbrand 250 ml瓶(目录号:FB-800-250);VWR 500 ml瓶[硼硅酸盐3.3;目录号:215-1594);Scott Duran 1000 ml瓶(原GL 45系列)]
  6. pH计(用于调节溶液的pH值)

程序

样品处理的一般注意事项:>

  1. 样品固定最好在密封的无核糖核酸酶容器中进行,如Eppendorf或Falcon管。
  2. 一旦样品再水合,它们就可以转移到无rnase的原位杂交篮中(如图1a所示)。
  3. 样品的密度可能会影响原位杂交结果:过多的小瓶/篮通常会产生染色不良/损坏的样品,过少的样品(例如,一个胚胎)可能会过度染色(可能是由于探针/靶点比例不平衡造成的)。

  1. 样品固定/脱水(表1)
    注:
    1. 应根据实验室安全指南处理和处置固定剂。
    2. 固定(固定剂的选择、固定时间)必须适应样本类型。
    3. 提供了两种可供选择的固定方式,用于比色原位杂交(CISH)和荧光原位杂交(FISH)。>

      表1。固定a


  2. 再水化(表2)

    表2。再水化

  3. (可选)>渗透性;蛋白酶k处理渗透性组织(表3)
    注:
    1. 每批新酶的处理时间应滴定,因为强度可能不同。一个有用的经验法则是对测试样本进行处理,直到出现组织损伤,并将结果时间减半(有关更多详细信息,请参见注释部分的图3)。
    2. 这一步骤对于非常脆弱的组织来说可能是不必要的。

    >表3。渗透性

  4. (可选)乙酰化(表4)
    注意:由于探头与带正电表面结合,此步骤可能有助于减少非特定信号。>

    表4。乙酰化

  5. (可选)重新固定(表5)
    注:
    1. 根据样本类型(例如脆弱的无脊椎动物胚胎),可能需要在杂交之前进行额外的再固定步骤。
    2. 一般来说,蛋白酶K处理后最好重新固定。
    3. 戊二醛可以省略:建议在第一次设置方案时测试几个选项。戊二醛渗透组织的速度较慢,对于厚度小于1 mm的样品,戊二醛的使用更为可取(Eltoum等人,2001年)。

    表5。重新固定


  6. 杂交(表6)
    注:
    1. 在杂交前/杂交阶段不要让温度下降。转移吊篮或增加探头可能需要很长时间时间。处理多个样本时,请设置可管理的批处理。
    2. 尿素基杂交缓冲液往往是粘性的,蒸发可能是一个重要问题,导致晶体的形成,将损害样品。小心密封小瓶/多孔板(图1c)。
    3. 注意,在加热的杂交缓冲液中,一些样品类型可能会变得暂时透明。在这个阶段,它们也会更脆弱。>

    表6。杂交

    表6。继续



  7. 阻塞(表7)

    表7。阻塞

  8. 信号检测
    AP抗体,比色反应
    表8。比色信号检测


    表8。继续


    荧光检测(pod抗体,信号放大)>(表9)

    表9。荧光信号检测


  9. CISH安装(表10)

    表10。比色样品-安装

  10. 鱼架(表11)

    表11。荧光样品-安装

数据分析

尿素变异体原位杂交方案产生了可靠的结果。样本形态保存良好,良好的信号背景比允许细胞分辨率检测基因表达。图2描述了在aclytiamedusa上执行的比色(cish)和荧光(fish)方案的结果。
关于甲酰胺和尿素方案的进一步比较,请参考原始论文(Sinigalia等人,2018年),例如来自不同物种的样本,以及结果的更详细分析。


图2。水母中基因表达的检测。rfamide>(clytia>基因的登录号:kx496951)在神经系统细胞中表达。尿素原位杂交法检测散布在水母体内的细胞,包括(从左到右):触手、触角球茎、伞周的圆形管和伞。组织保存完好,特别是脆弱的伞。CISH的结果是,焦点集中在触手和圆管中的散在细胞上。用蔡司Axio成像仪A2拍摄的图像。b.FISH结果,用徕卡SP8共焦显微镜拍摄的Z叠加图像的最大投影(用斐济完成)。两幅图像均被裁剪;比例尺=50μm。

笔记

  1. 控制
    1. 强烈建议同时进行阴性对照(例如,>,使用感测探针,或对未在所研究样本中表达的基因使用长度相似的探针)和阳性对照(使用可靠的已知反义探针),特别是在测试任何新探针或可选探针时步骤。
    2. 当首次测试这种基于尿素的方案时,建议与最终以前的基于甲酰胺的方案(如果已经在实验室中使用)进行平行的原位杂交。

  2. mrna探针
    1. 核糖探针(mrna探针)可以由线性化的模板质粒或pcr产物合成。原始研究论文(Sinigalia等人,2018年)中所示的DIG标记的RNA探针,包括本方案中使用的rfamide>探针,是使用Promega的其中一个RNA聚合酶试剂盒(T7、SP6或T3多聚酶)合成的。插入)。对于探针合成,按顺序添加:2μl 10x dig标记混合rna(roche);0.5μl rnasin核糖核酸酶抑制剂(promega);2μl 100 mm dtt(promega);4μl转录优化5x缓冲液(promega);1-2μg dna模板(计算适当体积);无rnase h2o至20μl总体积;0.5μl t7/sp6/t3 rna聚合酶(promega)。在37°C下反应2-5小时,然后用1.5μL RQ1无核糖核酸酶DNA酶(Promega)在37°C下消化DNA模板30分钟,然后加入30μL无核糖核酸酶H2O洗脱,用Illustra Probequant G-50微柱纯化(GE Healthcare)。通过在1%琼脂糖凝胶上运行1μL纯化探针,用纳米滴法定量检测探针完整性。探针可以储存在-20°C或-80°C,添加50%(v/v)无核糖核酸酶的甲酰胺有助于保持核糖核酸的完整性。
    2. 建议始终合成反义(与mrna互补)探针和感测探针,作为结合特异性的对照。
    3. 探针通常是根据基因的编码序列设计的,但它们也可以针对3’utr区域(这可能有助于区分高度相似的基因)。从cdna中克隆所需区域,可能来源于从感兴趣的阶段/身体区域提取的mrna(不同阶段/组织可能表达同一基因的不同剪接形式)。
    4. 建议探头长度在500到800之间,最大为1500 bp。长探针可以通过碱性水解而破碎,例如>。
      碱性水解(Cox等,1984):通过添加1:1体积的水解缓冲液(40 mm NaHCO3/60 mm Na2Co3,pH 10.2)新鲜水解探针在无核糖核酸酶的水中制备。在60℃下孵育一段时间(以分钟表示),计算如下:


      其中,l0是原始转录长度(以kb表示),lf是所需片段长度(以kb表示)。
      加入醋酸钠(0.1 m终产物,pH6.0)和冰醋酸(0.5%,v/v)中和,用乙醇沉淀。

  3. 调整协议/变体
    1. 该方案基于原位杂交(sinigagliaet al>,2018年),但其目的是代表一般准则。当它适应新的研究有机体/组织/细胞时,建议首先用尿素溶液取代原始杂交缓冲液中的甲酰胺,最终浓度为4 M。
    2. 方案的变体是可能的,特别是荧光原位杂交已经成功地与免疫染色或edu反应耦合(数据未显示)。
    3. 该方案也可用于组织切片(冷冻或石蜡包埋),在这种情况下,可能需要进一步调整(例如>,降低洗涤剂或杂交缓冲液中的尿素浓度)。
    4. 尿素基杂交缓冲液也可以用SMFISH进行检测。
    5. 蛋白酶k治疗是可选的。蛋白酶K能消化蛋白质,从而使组织渗透,并有助于探针和试剂的渗透。治疗过强可能损害组织形态,而消化不足可能导致信号检测不良。每批新酶的培养时间和酶浓度都应进行评估。


      图3。蛋白酶k处理的滴定法。图像显示10μg/ml蛋白酶k在1x pbst中对固定的水母进行消化。治疗30分钟后开始出现可见损伤(黄色星号),40分钟后水母严重受损(注意绒毛和性腺分离)。根据指导方针根据主要方案(见表3),蛋白酶K处理应在原位杂交期间持续15-20分钟。

  4. 故障排除
    1. 损坏样品
      1. 确保样品始终被溶液覆盖,不要让它们干燥。
      2. 确认杂交缓冲液没有蒸发。
      3. 再水化后,在杂交前加入第二个固定步骤。
      4. 去除渗透步骤,或最终缩短治疗时间。
      5. 小心处理样品和篮子。样品在甲醇和杂交溶液中可能更易碎。
      6. 如果损坏是由于在样品上形成晶体造成的,则尝试使用milliq h2o快速清洗样品。
    2. 无信号/信号差
      1. 验证探针质量(检查降解情况)并使用正确的反义探针。长探针通常表现较差,但最佳长度是物种和基因特异性(目标是500-800 bp)。探针可以通过碱性水解而破碎(例如>,见注释部分的B4点);这将提高对目标的穿透力和识别能力。
      2. 添加渗透性步骤/重新测试/更新蛋白酶K批次。
      3. 增加/减少杂交混合物中的探针浓度。
      4. 在CISH的情况下:延长信号检测步骤(经常更新NBT/BCIP混合,同时将样品保持在4°C)。如果是鱼:延长信号检测反应,或重复。
      5. 对于CISH:使用新制备的染色液。新制备的NTMT(如果超过一天请勿使用,在不使用时保持在4°C)。
      6. 如果是鱼:在使用前准备好缓冲液。
      7. 增加/减少杂交混合物中的尿素浓度(8 m至2 m):一般来说,较高浓度将具有更强的渗透效果,建议用于难处理/硬组织。
      8. 验证溶液的pH值,特别是茶和SSC储备溶液的pH值。
      9. 样本可能是固定的,核实一下。最好去除戊二醛,以防将其添加到固定液中。

    3. 非特定信号
      1. 对于CISH:将左旋咪唑添加到NTMT和染色混合物中。
      2. 对于CISH:在NBT/BCIP显色反应之前,延长与NTMT的孵育时间。
      3. 对于CISH:保护NBT/BCIP混合物不受光照。验证储备溶液没有变紫或变黑,并且看起来是均匀的。
      4. 在CISH的情况下:试着进行鱼类替代(允许更精确的观察,避免非特定沉淀物沉积的最终问题)。
      5. 通宵预杂交。
      6. 加入乙酰化步骤。
      7. 确保醋酸酐溶液(步骤d中的步骤1和2)得到有效混合,并且样品与之接触良好。
      8. 提高杂交温度。
      9. 降低探针浓度。循环探针(将杂交混合物储存在-20°C下)。
      10. 增加杂交后洗涤的持续时间/强度。通过改变ssc缓冲液的浓度来调节严格性(低严格性为5-6x,高严格性为2-0.1x)。
      11. 在开始显色反应之前,确保样品上没有灰尘/污垢:颗粒可能来自回收篮的洗涤不足、溶液(过滤它们,特别是NTMT溶液)、衣服。如果存在,请手动移除它们。
        一般来说,附着在样品上的微粒通常来自于旧溶液:更新它们,特别是染色剂,并精确地调节pH值。在染色前加入ntmt减去缓冲液(过滤)也可能有帮助。
      12. 对于CISH:扩展程序H(比色检测)和程序I中的步骤9(CISH安装),将样品保持在4°C。
      13. 验证溶液的pH值,特别是茶和SSC储备溶液的pH值。

    4. 信号太强/太快
      1. 降低探针浓度。
      2. 对于CISH:稀释染色液。减少染色时间。在染色液中加入左旋咪唑和/或在4℃下进行染色反应,以减缓AP反应。
      3. 在CISH的情况下:试着进行鱼类替代(允许更精确的观察,避免非特定沉淀物沉积的最终问题)。
      4. 增加水洗的松紧度。
      5. 加入乙酰化步骤。

食谱

  1. 10x PBS(pH 7.4)储备溶液
    1. 称取25.6g Na2HPO4·7h2O,80g NaCl,2g KCl,2g KH2Po4
    2. 用毫厘H2O调高到1升
    3. 高压灭菌器
    4. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  2. 1x PBST
    1. 在1X PBS溶液中加入0.1%吐温-20
    2. 在室温下储存,如果出现沉淀物/颗粒(瓶子可能已被污染),则丢弃
  3. 下摆缓冲
    0.1 mHepes,pH值6.9
    50毫米egta,ph值7.2
    10毫米mgso4
    新鲜的准备
  4. cish固定缓冲液
    3.7%甲醛加
    0.4%戊二醛
    1x PBS(pH 7.4)
    新鲜的准备
  5. 鱼固定缓冲液
    3.7%甲醛缓冲液
    新鲜的准备
  6. 脱水缓冲液
    50%1倍PBST
    50%甲醇
    新鲜的准备
  7. 补液缓冲液
    50%甲醇
    50%1倍PBST
    新鲜的准备
  8. 渗透缓冲区
    1X PBST中10μg/ml蛋白酶K
    新鲜的准备
  9. 中和缓冲液
    1X PBST中2 mg/ml甘氨酸
    新鲜的准备
  10. 10倍茶原液
    1. 在H2O中制备1m溶液
    2. 将pH值调整到7.8
    3. 不要高压灭菌
    4. 小心喝茶(不要摇晃)
    5. 在室温下储存,如果出现沉淀物/颗粒(瓶子可能已被污染),则丢弃
  11. 0.1米茶叶
    用1X PBST稀释10X茶原液至0.1 M三乙醇胺(茶)
    新鲜的准备
  12. 0.25%醋酸酐在0.1 m茶叶中
    将0.25%醋酸酐(v/v)加入0.1 m茶溶液中,充分混合
    新鲜的准备
  13. 再固定缓冲液
    3.7%甲醛加0.2%戊二醛,1X PBST
    新鲜的准备
  14. 8 m尿素储备溶液
    1. 将尿素溶解在毫克H2O中,计算所需量(mW=60.06 g/mol)
    2. 注意反应是吸热的
    3. 新鲜制备,必要时过滤消毒
    4. 该溶液可用于杂交和杂交后步骤,在这种情况下,保持在4°C
  15. 20x SSC(柠檬酸盐)储备缓冲液(1L)
    注:20倍浓度的缓冲液可防止真菌和细菌生长。>
    1. 将175.3 g NaCl和88.2 g柠檬酸三钠(Na3C6H5O7)溶于900 ml毫质量H2O中。
    2. 将pH值调整到7.0,顶部调整到1 L,并使用毫质量H2O
    3. 高压灭菌器
    4. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  16. 20%十二烷基硫酸钠 在低温下可能发生沉淀。在这种情况下,使用前先让缓冲区预热
  17. 尿素基杂交缓冲液
    最终浓度:
    5x子系统控制器
    1%右旋糖酐
    4 M尿素
    50μg/ml肝素
    50μg/ml的trna(或鲑鱼精子)
    1%十二烷基硫酸钠
    准备(10毫升)按以下顺序添加,新鲜准备:
    2.5毫升20倍ssc
    0.1g右旋糖酐粉末(溶解良好)
    5ml 8m尿素储备溶液
    25μl肝素(20 mg/ml储备)
    10μl trna
    5.1毫升毫克H2O
    500μl 20%十二烷基硫酸钠
    注:
    1. 注意,过早添加十二烷基硫酸钠可能会导致出现沉淀。
    2. 不要剧烈搅动,以免产生气泡。
    3. 杂交缓冲液也可以在-20℃下校准和储存,在这种情况下避免多次解冻/冷冻循环。
  18. 50%杂交缓冲液
    50%尿素基杂交缓冲液
    50%1倍PBST
    新鲜的准备
  19. 清洗i
    最终浓度:
    4 M尿素
    0.1%吐温-20
    5x子系统控制器
    将milliq h2o加到体积中
    新鲜制备;或在使用前不久(如使用前一晚)
  20. 清洗II
    最终浓度:
    2m尿素
    0.1%吐温-20
    2x子系统控制器
    将milliq h2o加到体积中
    准备新鲜的;或不久前(例如,使用前一晚)
  21. 清洗III
    最终浓度:
    0.1%吐温-20
    2x子系统控制器
    将milliq h2o加到体积中
    新鲜制备;或在使用前不久(如使用前一晚)
  22. 10x单克隆抗体缓冲储备溶液
    1. 加入700毫升的毫质量氢2o:116克马来酸、87克氯化钠、40克氢氧化钠
    2. 小心地将pH值调整到7.5,并用毫厘H2O加满到1升
    3. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  23. 1x mAbt缓冲器
    1. 以1:10稀释储备溶液,用毫厘H2O,加入0.1%吐温-20
    2. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  24. 10x封闭试剂储备溶液
    1. 在马来酸缓冲液中溶解至最终浓度10%(w/v),摇动并加热至60°C
    2. 原液应蒸压、校准并储存在-20°C下
  25. 1X闭锁缓冲器
  26. 5 m氯化钠
    1. 将292 g NaCl溶于800 ml的Milliq H2O
    2. 用毫厘H2O将音量调整到1L
    3. 灭菌用高压灭菌器
    4. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  27. 1 M Tris HCl,pH 9.5
    1. 将121.1g Tris碱溶于800ml的Milliq H2O中
    2. 加入HCl调节pH至9.5(使溶液达到室温以精确调节pH)
    3. 用毫厘H2O将音量调整到1L
    4. 灭菌用高压灭菌器
    5. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  28. 1米mgcl2
    1. 将203.3g mgcl2·6h2o溶解于800 ml的milliq h2o中
    2. 调节音量至1升,毫质量h2o
    3. 高压灭菌器
    4. 室温储存,如果出现沉淀物/颗粒,则丢弃(瓶子可能已被污染)
  29. ntmt缓冲区
    最终浓度:
    100毫米氯化钠
    100毫米Tris HCl
    50 mm的mgcl2
    对于100毫升,添加:>
    2毫升5 M氯化钠
    10毫升1 M Tris HCl(pH 9.5)
    5毫升1米氯化镁2
    1毫升吐温-20
    毫厘h2o体积
    新鲜的准备
  30. ntmt减去缓冲区
    最终浓度:
    100毫米氯化钠
    100毫米Tris HCl
    对于100毫升,添加:>
    2毫升5 M氯化钠
    10毫升1 M Tris HCl(pH 9.5)
    1毫升吐温-20
    毫厘h2o体积
    新鲜的准备
  31. ntmt缓冲区中的nbt/bcip
    1. 添加到ntmt缓冲区:
      0.08 mg/ml硝基蓝四氮唑
      0.1 mg/ml 5-溴-4-氯-3-吲哚基磷酸盐
    2. 新鲜准备,避光

致谢

我感谢卢卡斯·莱克莱尔和伊夫林·霍利斯顿对手稿和讨论的评论,并感谢霍利斯顿/莫莫斯小组的其他成员,特别是桑德拉·切瓦利埃的讨论和协助。我也要感谢所有在他们的研究组织上测试这个方案的人,特别是冈萨洛·基罗加·阿蒂加斯、洛伦佐·里奇、丹尼尔·蒂尔。我感谢匿名评论者对手稿的有益评论。我也感谢恩里克·阿尔伯莱达对手稿的最终版本发表评论。资金来源:国家可再生能源机构,批准“Medusevo”ANR-13-PDOC-0016。

相互竞争的利益

提交人声明没有相互竞争的利益。

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引用:Sinigaglia, C. (2019). A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol. Bio-protocol 9(17): e3360. DOI: 10.21769/BioProtoc.3360.
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