Oneill Lab:WMISH Protocol

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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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

1. Dissect embryos into PBS
2. Rinse serially in clean PBS
3. Fix embryos in 20 volumes of fresh 4% Paraformaldehyde PBS
   • Fix overnight at 4°C
4. Quickly rinse embryos in PBS
5. Wash for 10 minutes in fresh PBS
6. 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
7. Store embryos in 100% MeOH at -20°C

Day II: Pretreatment and Hybridization

1. Rehydrate embryos along methanol row
   • 75% MeOH / 25% PBST
   • 50% MeOH / 50% PBST
   • 25% MeOH / 75% PBST
   • 15 minutes shaking for each solution
2. Wash 3x minutes in PBST
3. Treat with ProK (30μg/mL) at 37°C (See Table Below)
   • Dilute ProteinaseK stock (20 mg/ml) with ProK Buffer
4. Rinse 3x PBST
5. Wash 15 minutes PBST
6. Post-fix 30 minutes at 4°C
7. Rinse 3x PBST
8. Wash 2x 15 minutes PBST
9. Rinse in 1:1 PBST:Hybridization Buffer
   • Allow embryos to settle, typically 1-2 minutes
10. Rinse with hybridization buffer
    • Allow embryos to settle
11. Prehyb for 2 hours at 70°C
12. Incubate embryos in hybridization buffer + DIG-labeled RNA probe at 70°C overnight
    • Use 2.4 μL probe / mL hyb buffer

Proteinase K Treatment

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

1. Rinse 2x with 70°C Hybridization Buffer
2. Wash 2x 30 minutes with 70°C Hybridization Buffer
3. Wash 1x 30 minutes with 70°C 1:1 Hyb:TBST
4. Rinse 2x room temp TBST
5. Wash 2x 30 minutes TBST

Antibody Hybridization

1. Rinse 2x MABT
2. 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
3. Shake overnight at 4°C in blocking buffer with anti-DIG AP conjugated antibody
   • Use 1μL antibody / ml blocking

Day IV

Antibody washes

1. Rinse 3x in MABT
2. Use fine forceps to pierce holes n the head to alleviate ab build-up
3. Wash all day in MABT, changing once per hour
4. Continue to wash in MABT
   • 3 days changing wash once per day
   • Or one day changing wash every hour

Detection

1. Wash 2x 20minutes in NTMT (Genius III+10% Tween20)
2. Incubate in the dark in BCIP/NBT
   • Use 16μL detection mix / 3 ml NTMT
3. To stop reaction, wash 3x 10 minutes in PBST
4. Store embryos in PBST + 1mM EDTA + 0.1% Sodium Azide (!) at 4°C

Links and Contacts

Seth Kasowitz [1]

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