RNA Whole Mount In Situ Hybridization
Chick Embryos
Embryo preparation
 Fix embryos in fresh 4% paraformaldehyde in PBS (4% PFA) at 4oC overnight
 Dissect embryos into PBT, and wash once in PBT for 5 minutes, gently rocking
 Dehydrates embryos in methanol using a graded methanol/PBT series (25%, 50%,
75% and 100% methanol). Allow embryos to rock gently at RT for 5 minutes in
each wash solution
 Do one additional wash in 100% methanol, 5 minutes. Embryos can be stored at
–20oC (use either glass scintillation vials or plastic screw-cap tubes).
Day I: Prehybridization and Hybridization
 Rehydrate samples in a 75%, 50%, 25% methanol/PBT series.
 Wash 2X in PBT for 5 minutes at RT
 Bleach embryos (not early stages < st 16) with 6% hydrogen peroxide in PBT for
1 hour at RT with gentle rocking. You can reduce or extend bleaching time if
desired. Embryos MUST have been previously dehydrated in methanol if you are
going to include the bleaching step!!
 Wash 3 times with PBT, 5 minutes each
 Proteinase K treatment: a 10mg/ml stock aliquots are stored at –20oC
Make stage appropriate dilution for Prot K in PBT (see below)
Times can be critical
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Stage
[Proteinase K]
< st 10
st 10-st 18
st 18-st 24
st 26-27
st 28-29
st 30-31
st 32 and older
1-3 ug/ml
10ug/ml
10ug/ml
20 ug/ml
30 ug/ml
40 ug/ml
50 ug/ml
Time
15 minutes RT
15 minutes RT
20 minutes
15 minutes
15 minutes
15 minutes
15 minutes
Immediately postfix in 4%PFA and 0.2% gluteraldehyde in PBT, 20 minutes RT
Wash 3X for 5 minutes each in PBT
Incubate at 70oC in hybridization solution (WM in –20oC) for 1hr. Pre-hybe in
1ml volume or until embryos are just covered in solution.
Replace hybridization solution with fresh, add RNA probe (usually 10-20ul of a
transcription reaction). You can also re-heat previously used probes in hybe
buffer. Incubate overnight at 70oC gently rocking.
Day II: Post-hybridization washes, blocking, and antibody incubation
 Prewarm solution I and III (stored at –20oC) at 70oC
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Wash embryos 3X for 30 minutes each at 70oC in prewarmed solution I
Washing embryos 3X for 30 minutes each at 65oC in prewarmed solution III
Wash 3X with fresh TBST for 5 minutes each @ RT
Blocking step: incubate in 10% heat-inactivated sheep serum in TBST for at least
1 hour at RT
Remove block solution. Add antibody solution and incubate overnight at 4oC
o Ab solution: 1:5000 anti—DIG-AP antibody in 10% h-i sheep serum in
TBST
Day III: Post-Antibody Washes
 Wash 3X for 5 minutes each with TBST @ RT
 Wash 5X for 1-1.5hr in TBST @ RT
 Wash overnight in TBST @ 4oC, rocking gently