A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol

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.

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