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
 * 4) *Fix overnight at 4°C
 * 5) Quickly rinse embryos in PBS
 * 6) Wash for 10 minutes in fresh PBS
 * 7) Serially dehydrate along methanol row
 * 8) *25% MeOH / 75% PBST
 * 9) *50% MeOH / 50% PBST
 * 10) *75% MeOH / 25% PBST
 * 11) *100%MeOH
 * 12) *PBST is PBS with 0.1% Tween20
 * 13) *Incubate in each solution at room temperature shaking for 15 minutes
 * 14) Store embryos in 100% MeOH at -20°C

Day II: Pretreatment and Hybridization

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

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
 * 3) *Blocking buffer is 10% heat-inactivated sheep serum in 2% blocking solution with 0.1% Tween20
 * 4) *To inactivate sheep serum, heat to 55°C for 30 minutes then cool on ice and store at -20°C
 * 5) Shake overnight at 4°C in blocking buffer with anti-DIG AP conjugated antibody
 * 6) *Use 1μL antibody / ml blocking

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
 * 5) *3 days changing wash once per day
 * 6) *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
 * 3) *Use 16μL detection mix / 3 ml NTMT
 * 4) To stop reaction, wash 3x 10 minutes in PBST
 * 5) Store embryos in PBST + 1mM EDTA + 0.1% Sodium Azide (!) at 4°C

Links and Contacts
Seth Kasowitz [mailto:seth.kasowitz@uconn.edu]

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