Burdine Lab - In situ checklist

We use this checklist to monitor our in situs and paste into our lab notebooks to record the experimental details and deviations.

In situ protocol: Checklist

Day 1

-5’ in 75% MeOH: 25% PBT

-5’ in 50% MeOH: 50% PBT

-5’ in 25% MeOH: 75% PBT

-5’ in PBT

-Can treat embryos with peroxide to remove pigment at this step

(-5’ in PBT – only if peroxide treated)

-ProK use appropriate time for stage of embryos (5', 10',20')

-20’ 4% PFA

-5’ in PBT

-2h in HYB - 68˚C

-O/N (12-18h) HYB plus probe - 68˚C

Day 2

Following at 70^oC:

-10’ in 75% HYB: 25% 2x SSC

-10’ in 50% HYB: 50% 2x SSC

-10’ in 25% HYB: 75% 2x SSC

-10’ in 2x SSC

-30’ in 0.2x SSC

Following at RT:

-5’ in 75% 0.2x SSC: 25% PBT

-5’ in 50% 0.2x SSC: 50% PBT

-5’ in 25% 0.2x SSC: 75% PBT

-5’ in PBT

- OPTIONAL incubate embryos 45’ in 0.1M Glycine pH2.2 + 0.1% Tween (to remove endogenous AP)

- 5’ PBT

- block 2h 2mg BSA/ml PBT + 5% sheep serum on rocker at RT

-O/N at 4˚C in preabsorbed antibody (200ul of 1:2000)

Day 3

Following at RT (2mg BSA/ml PBT steps on a rocker):

-15’ in 2mg BSA/ml PBT

-5’ NTMT

-develop with NBT/BCIP

-stop with 3x NTMT rinses

-Rinse 1x in PBT

-Fix O/N in 4% PFA