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Tissue Process for Whole Mount In Situ

*The following wash and dehydration steps should be carried out at 4 °C on a shaker.( No shaking with Day 8.5-9.5 embryos )

*Use all RNase free solutions.

1. Fix embryos overnight in 4% paraformaldehyde/PBS at 4°C with shaking.
2. Wash with 1min PBT(PBS+0.1%Tween-20).
3. Wash 3X 10 min with PBT at 4°C.
4. Dehydrate :
   25% methanol in PBT 20-40min.
   50% methanol in PBT 20-40min.
   75% methanol in PBT 20-40min.
   100% methanol in PBT 20-40min.
   
   Change to new methanol.
   
   
5. Store at -20°C until further use.

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