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In situ hybridization zebrafish embryo prep - paraformaldehyde damage embryos over time??? (Jul/27/2007 )

I throw my embryos in 4% paraformaldehyde overnight right away, then dechorionate them and store at -20 in methanol. However, I have these old embryos in 4 deg C in 4% PFA that have been there like a month (just wild type so didnt need that many at the time). Does having it in the PFA too long affect the in situs? Should I just throw them away or are they still good if I dechorionate and store them in MeOH -20?

Thanks for any tips.



Normally I don't use embryo which is fixed more than 24 hpf as i read somewhere, too long in PFA is not good for embryos......


What specifically does it do after too long? Any photos of in situs done comparing normal overnight 4%PFA fixation versus longer (days or weeks)?

Also, does it make a difference if you fix them while the chorions are still on and then immediately transfer them to methanol when you dechorionate them or should you keep them in paraformaldehyde longer after you dechorionate them?