This protocol is designed for high-sensitivity single-gene in situ hybridization in Helobdella embryo, using chromogenic AP substrate BM Purple or NBT/BCIP. Its performance is fairly consistent and is suitable for screening/survey and in experimental conditions.

Day 0: Fixation

1. For embryos of late stage 9 or older, manually remove vitelline membrane with tweezers and bath embryos in ice-cold relaxation solution before fixation.
2. Fix embryos with 4% formaldehyde in diluted PBS (0.25X for stages 1-6; 0.5X for stages 7-11) for 1hr at room temperature (r.t.) or overnight at 4°C.
3. Wash embryos with 1XPBS for several times.
4. For stages 1-9, remove vitelline membrane using broken glass pipette.
5. 1 wash in PBS:MeOH (1:1) and 3 washes in 100% MeOH. Store embryos in MeOH at -20°C for at least overnight.

Day 1: Pretreatment (for stage 1-9, ok to skip steps 3 for most probes)

1. Wash embryos with PBS:MeOH(1:1), then 3X brief washes with PBS + 0.1% Tween (PBT).
2. Add Proteinase K solution to a final concentration of 20 μg/ml in PBT; i.e. 1 microliter 20 mg/ml stock to 1ml PBT to the embryos.
3. Contrary to conventional wisdom, proteinase K treatment is not essential for probe penetration; rather, it reduces background by facilitating probe escape during stringency wash. For stages 1-8, effect of proteinase treatment is negligible; OK to skip proteinase treatment; 1 minute treatment at r.t. effectively cut all background staining. For stage 9, 5 minute at r.t. produces good results. For special purposes (e.g. combining Ab staining), skip proteinase K treatment. For stage 10, 15 minutes at r.t. is required. For stage 11 and juvenile, 30 minutes at r.t. is required. Not possible to skip proteinase K treatment for stages 10-11 embryos.
4. Remove Proteinase K solution, and wash twice with PBT.
5. Post-fix embryos with 4% formaldehyde in 0.75XPBT for 20-30 min at room temperature. 5X washes with PBT.
6. (optional, only needed if background is high or when using TSA) Acetylation of reactive amine by acetic anhydride treatment. This treatment may also reduce nonspecific probe binding. To do this, wash embryos briefly in 0.1 M triethanolamine (pH 8). Replace the solution and incubate the embryos in 1 ml of 0.1 M triethanolamine at r.t. for 5 minutes. Add 3 μl of acetic anhydride to 1 ml of 0.1 M triethanolamine solution with embryos in it. Mix the solution by gentle rocking for 5 minutes. Add another 3 μl of acetic anhydride. Incubate on rocking table for another 5 minutes. Wash 3X with PBT briefly.
7. Transfer the embryos into 1.5 ml tubes.
8. Add 50 μl of PreHyb:PBT (1:1) to the embryos and wait until embryos settling down.
9. Wash embryos twice with 100 μl PreHyb.
10. Replace the solution with 200 μl PreHyb, and incubate at hybridization temperature (65-70°C) for overnight.

Day 2: Hybridization

1. Add probe to 200 μl fresh PreHyb buffer; the amount of probe is determined empirically for each batch of probe and each stage. Start with ~1 ng/μl (or 1/100 dilution from the stock) for stages 1-9 and 0.25 ng/μl (or 1/400 dilution from the stock) for embryos of stages 10-11. Note that BM Purple staining is more sensitive to probe overdose than NBT/BCIP, and thus one should be more careful with probe concentration when using BM Purple.
2. (optional) Carrying out hybridization in PreHyb-DS instead of PreHyb increases sensitivity.
3. Place the probe solution in the oven to reach hybridization temperature.
4. Remove PreHyb solution from the embryo and save it for the following washing step. Add the pre-warmed probe solution and hybridize for 24-48 hrs.

Day 3/4: Probe Removal

1. Wash with warm PreHyb (~200 μl each, saved from the blocking step) in the oven for 10 min.
2. Wash the embryos with warm 2XSSC (0.5-1 ml each tube; may add 0.05% CHAPS to prevent embryos from sticking to the tubes) in the oven for 20 min.
3. Wash the embryos twice with warm 0.2XSSC (0.5-1 ml each tube; 0.05% CHAPS, optional) in the oven for 20 min each.
4. Wash the embryos twice with warm 0.1XSSC (0.5-1 ml each tube; 0.05% CHAPS, optional) in the oven for 20 min each.
5. Remove as much liquid as possible, and then allow the tube and embryos to return to r.t. Rinse embryos in PBT twice, followed by a 5 min wash in PBT.

Day 3/4: Antibody Labeling

1. Transfer the embryos into 0.6 mls tube using flamed glass pipet. Remove as much liquid as possible; then add a desirable amount of Ab blocking solution. Block the embryos in room temperature on a rocking table for 1 hour.
2. Add 1/2500 anti-dig antibody (Roche 11093274910; diluted 1:1 with glycerol for long-term -20°C storage) to blocking solution.
3. Incubate overnight on a rocking table at 4°C.

Day 4/5: Antibody Wash

1. Remove the Ab solution; rinse the embryos 3X with PBT; 1 min each. Wash the embryos with PBT for 3-4 hours; change PBT every 30 min.
2. Proceed to color reaction with either NBT/BCIP or BM Purple. See additional information about the choice of the chromophore.

Recipes

• Relaxation solution: 4.8 mM NaCl, 1.2 mM KCl, 10 mM MgCl₂, 8% EtOH
• 20X SSC: 3 M NaCl, 0.3 M trisodium citrate, pH 7.0
• 50X Denhardt's Solution: 1% Ficoll 400, 1% polyvinylpyrrolidone, and 1% bovine serum albumin
• PreHyb: mix 25 mg torula RNA type VI (Sigma R6625) into 25 ml deionized formamide (Life Technologies/Ambion AM9342 or Sigma F9037); add 12.5 ml 20X SSC, 0.25 ml 10 mg/ml heparin, 1 ml 50X Denhardt’s Solution, 0.5 ml 10% Tween-20, 0.46 1 M citric acid; bring to 50 ml with ddH₂O
• PreHyb-DS (PreHyb + 5% dextran sulfate): mix 25 mg torula RNA type VI (Sigma R6625) and 2.5 g dextran sulfate (Sigma D8906) into 25 ml deionized formamide (Life Technologies/Ambion AM9342 or Sigma F9037); add 12.5 ml 20X SSC, 0.25 ml 10 mg/ml heparin, 1 ml 50X Denhardt’s Solution, 0.5 ml 10% Tween-20, 0.46 1 M citric acid; bring to 50 ml with ddH₂O.
• Ab block solution: 10% Roche Western Block Solution (and 1% normal sheep serum, optional, if Roche's sheep anti-dig AP conjugate is used), 0.1% Tween-20 in PBS

Related protocols

• Probe synthesis
• Double in situ hybridization (with chromogenic color reaction)
• Multiplex fluorescent in situ hybridization