...
In situ protocol: Checklist
Day 1
-5' 5’ in 75% MeOH: 25% PBT
-5' 5’ in 50% MeOH: 50% PBT
-5' 5’ in 25% MeOH: 75% PBT
-5' 5’ in PBT
-5' 5’ in PBT
-5' 5’ in PBT
-5' 5’ in PBT
-Can treat embryos with peroxide to remove pigment at this step
(-5' 5’ in PBT – only if peroxide treated)
(-5' 5’ in PBT – only if peroxide treated)
-ProK use appropriate time for stage of embryos (5', 10',20')
-20' 20’ 4% PFA
-5' 5’ in PBT
-5' 5’ in PBT
-5' 5’ in PBT
-5' 5’ in PBT
-5' 5’ in PBT
-2h in HYB - 68˚C
-O/N (12-18h) HYB plus probe - 68˚C
Day 2
Following at 70oC:
-10' 10’ in 75% HYB: 25% 2x SSC
-10' 10’ in 50% HYB: 50% 2x SSC
-10' 10’ in 25% HYB: 75% 2x SSC
-10' 10’ in 2x SSC
-30' 30’ in 0.2x SSC
-30' 30’ in 0.2x SSC
Following at RT:
-5' 5’ in 75% 0.2x SSC: 25% PBT
-5' 5’ in 50% 0.2x SSC: 50% PBT
-5' 5’ in 25% 0.2x SSC: 75% PBT
-5' 5’ in PBT
- OPTIONAL incubate embryos 45' 45’ in 0.1M Glycine pH2.2 + 0.1% Tween (to remove endogenous AP)
- 5' 5’ PBT
- 5' 5’ PBT
- 5' 5’ PBT
- block 2h 2mg BSA/ml PBT + 5% sheep serum on rocker at RT
...
Following at RT (2mg BSA/ml PBT steps on a rocker):
-15' 15’ in 2mg BSA/ml PBT
-15' 15’ in 2mg BSA/ml PBT
-15' 15’ in 2mg BSA/ml PBT
-15' 15’ in 2mg BSA/ml PBT
-15' 15’ in 2mg BSA/ml PBT
-15' 15’ in 2mg BSA/ml PBT
-5' 5’ NTMT
-5' 5’ NTMT
-5' 5’ NTMT
-develop with NBT/BCIP
...