Oneill Lab:WMISH Protocol

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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.


Contents

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)
  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]

O'Neill Lab Homepage

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