PECAM whole mount staining

PECAM whole mount staining

Embryos fix in either 4% PFA 1 hour, or Zn overnight.
Wash and store in 70% EtOH at 4C.

1.  Dehydrate 25%, 50%, 75%, 100%, 100% MeOH 15 min each RT (dilute MeOH with PBS)
2.  Incubate with 5% H2O2 in MeOH for 2hr at 4C, with rocking.
3.  Rehydrate 100%, 75%, 50%, 25% MeOH.  Wash in PBS + 0.1% Tx100 15 min.
4.  Wash in PBSST 1 hr 4C
5.  Incubate with PECAM (1:100 in PBSST) overnight 4C
6.  Wash in PBSMT 2 x 15 min RT, then 5 x 1 hr 4C
7.  Dilute biotinylated secondard 1:200 in PBSST.  o/n 4C
8.  Wash in PBSST 2 x 15 min RT, then 5 x 1 hr 4C
9.  Incubate with ABC-HRP in PBS.  After formed ABC complex, add NDS to 5%.  Add to samples, incubate o/n 4C.
10.  Wash in PBSST 2 x 15 min RT, then 5 x 1 hr 4C
11.  Final wiash in PBT
12.  Incubate embryos in DAB (no H2O2) RT 10 min.
13.  Incubate embryos in DAB + H2O2 RT 10 min.
14.  Rinse with PBS, then postfix with 4% PFA 1 hr RT or formalin.


PBSST = PBS, 5% NDS, 0.1% Tx100
PBSMT = PBS, 3% milk, 0.1% Tx100
PBT = PBS + 0.2% BSA, 0.1% tween 20
DAB = vector DAB kit NO Nickel!!!  Use virign plastic and special water.