Oneill Lab:WMISH Protocol
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Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology
Protocol for Whole-Mount in Situ Hybridization in mouse embryos. Based on protocol from Domingos Henrique and Davi Ish-Horowicz (ICRF Dev. Biol. Unit, Oxford). Modified from protocol of Ron Conlon, Phil Ingham, David Wilkinson, and Margaret Buckingham.
Day I: Dissection and Dehydration
- Dissect embryos into PBS
- Rinse serially in clean PBS
- Fix embryos in 20 volumes of fresh 4% Paraformaldehyde PBS
- Fix overnight at 4°C
- Quickly rinse embryos in PBS
- Wash for 10 minutes in fresh PBS
- Serially dehydrate along methanol row
- 25% MeOH / 75% PBST
- 50% MeOH / 50% PBST
- 75% MeOH / 25% PBST
- 100%MeOH
- PBST is PBS with 0.1% Tween20
- Incubate in each solution at room temperature shaking for 15 minutes
- Store embryos in 100% MeOH at -20°C
Day II: Pretreatment and Hybridization
- Rehydrate embryos along methanol row
- 75% MeOH / 25% PBST
- 50% MeOH / 50% PBST
- 25% MeOH / 75% PBST
- 15 minutes shaking for each solution
- Wash 3x minutes in PBST
- Treat with ProK (30μg/mL) at 37°C (See Table Below)
- Dilute ProteinaseK stock (20 mg/ml) with ProK Buffer
- Rinse 3x PBST
- Wash 15 minutes PBST
- Post-fix 30 minutes at 4°C
- Rinse 3x PBST
- Wash 2x 15 minutes PBST
- Rinse in 1:1 PBST:Hybridization Buffer
- Allow embryos to settle, typically 1-2 minutes
- Rinse with hybridization buffer
- Allow embryos to settle
- Prehyb for 2 hours at 70°C
- Incubate embryos in hybridization buffer + DIG-labeled RNA probe at 70°C overnight
- Use 2.4 μL probe / mL hyb buffer
Stage | Time (min) |
---|---|
E9.5 | 6.5 |
E10.5 | 8 |
E11.5 | 12 |
E12.5 | 25 |
E14.5 | 2x 20 |
Day III
Post-Hyb Washes
- Rinse 2x with 70°C Hybridization Buffer
- Wash 2x 30 minutes with 70°C Hybridization Buffer
- Wash 1x 30 minutes with 70°C 1:1 Hyb:TBST
- Rinse 2x room temp TBST
- Wash 2x 30 minutes TBST
Antibody Hybridization
- Rinse 2x MABT
- Incubate 2 hours at room temp in blocking buffer
- Blocking buffer is 10% heat-inactivated sheep serum in 2% blocking solution with 0.1% Tween20
- To inactivate sheep serum, heat to 55°C for 30 minutes then cool on ice and store at -20°C
- Shake overnight at 4°C in blocking buffer with anti-DIG AP conjugated antibody
- Use 1μL antibody / ml blocking
Day IV
Antibody washes
- Rinse 3x in MABT
- Use fine forceps to pierce holes n the head to alleviate ab build-up
- Wash all day in MABT, changing once per hour
- Continue to wash in MABT
- 3 days changing wash once per day
- Or one day changing wash every hour
Detection
- Wash 2x 20minutes in NTMT (Genius III+10% Tween20)
- Incubate in the dark in BCIP/NBT
- Use 16μL detection mix / 3 ml NTMT
- To stop reaction, wash 3x 10 minutes in PBST
- Store embryos in PBST + 1mM EDTA + 0.1% Sodium Azide (!) at 4°C