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
-5’ in PBT
-5’ in PBT
-5’ in PBT
-Can treat embryos with peroxide to remove pigment at this step
(-5’ in PBT – only if peroxide treated)
(-5’ in PBT – only if peroxide treated)
-ProK use appropriate time for stage of embryos (5', 10',20')
-20’ 4% PFA
-5’ in PBT
-5’ in PBT
-5’ in PBT
-5’ in PBT
-5’ in PBT
-2h in HYB - 68˚C
-O/N (12-18h) HYB plus probe - 68˚C
Day 2
Following at 70oC:
-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
-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
- 5’ PBT
- 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
-15’ in 2mg BSA/ml PBT
-15’ in 2mg BSA/ml PBT
-15’ in 2mg BSA/ml PBT
-15’ in 2mg BSA/ml PBT
-15’ in 2mg BSA/ml PBT
-5’ NTMT
-5’ NTMT
-5’ NTMT
-develop with NBT/BCIP
-stop with 3x NTMT rinses
-Rinse 1x in PBT
-Fix O/N in 4% PFA