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Chick Whole Mount
in
situHybridization
(Modified from
1.
Remove embryos from the egg;
dissectextraembryonic
membranes in Ringer's
Solution or calcium-free,
magnesium-free PBS.
2.
Fix embryos overnight at 4º C with freshly prepared 4%
paraformaldehyde (Fisher)
in PBS.
3.
Wash 2X (5 minutes per wash) at 4º C with
PBT.
Note:
all solutions are 10 ml/vial unless specified.
-Dehydrate
through ascending methanol series in PBT:
-25% MeOH (5 minutes)
-50% MeOH (5 minutes)
-75% MeOH (5 minutes)
-100% MeOH (5 minutes)
-100% MeOH (5 minutes)
- Store embryos in 100% MeOH
at -20º C until further use.
4.
Rehydrate through descending methanol series.
-75% MeOH (5 minutes)
-50% MeOH (5 minutes)
-25% MeOH (5 minutes)
-PBT (5 minutes)
-PBT (5 minutes)
5.
Bleach in 6% hydrogen peroxide in PBT (10
minutes).
6.
Wash 5X with PBT (5 minutes per wash).
7.
Permeabilize with Proteinase K (Boehringer Mannheim, 2
ug/mL) for 15 minutes (5
ml).
8.
Wash with 2 mg/ml glycine in PBT for 10 minutes.
9.
Wash 2X in PBT (5 minutes each).
10.
Fix embryos with 4% paraformaldehyde plus 0.2%
gluteraldehyde in PBT for 5
minutes).
11.
Wash 4X in PBT (5 minutes each).
12.
Wash 1X in prehybridization buffer (5 minutes, 5 ml).
- Incubate with fresh
prehybridization buffer for 1 hour at 65º-70ºC
(use hot water
bath).
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