Embryo Fixation (Timing: Overnight + 15 min)

  Fix dechorionated embryos of the appropriate developmental stage(s) in 4% (wt/vol) paraformaldehyde in 1 x PBS overnight at 4°C into Petri dishes. ( Paraformaldehyde solution should be not older than two days).

The next day, dehydrate the embryos in 100% methanol for 15 min at room temperature. I f the embryos were not treated with PTU during dechorionation step, remove pigmentation with H 2 O 2 as described in Box 1 prior to dehydration.

Place the eggs at -20°C in 100 % methanol for at least two hours before use.

( These embryos can be kept at  -20°C for several months).

Reagent Set Up:

4% (wt/vol) paraformaldehyde in 1 x PBS: Dissolve 4 g of paraformaldehyde in 1x PBS and heat until the powder has dissolved completely. When the powder is completely dissolved, cool the solution until it reaches room temperature (18 - 25°C). This solution, stored at room temperature, can be used for a couple of days.

Never boil the solution because paraformaldehyde will be degraded and the medium will acidify due to formation of formic acid.


(Adapted from Thisse Lab - In Situ Hybridization Protocol - 2010 Update)