2 replies to this topic

[missdaisy]

Hi all,

Maybe this is just a stupid question about the ISH, but I was just wondering.. let's say.. if I'm doing ISH on zebrafish embryo. After the rehydration stuff and proteinase K treatment, I'll need to fix the embryo with 4% paraformaldehyde and then wash them in PBST before incubate them with hybridization solution. If I fix the embryo with that paraformaldehyde but accidentally skip the washing step afterwards, will that interfere my ISH? If yes, how?

Thank you so much for all the answer!

Daisy

Edited by missdaisy, 25 April 2012 - 05:41 PM.

[bob1]

Potentially yes, the PFA can fluoresce, so if you are doing FISH this may be a problem.  For standard ISH, presence of PFA may alter the hybridisation temperature, and/or make the embryos hard to permeabilise.

[missdaisy]

bob1, on 26 April 2012 - 01:05 PM, said:

Potentially yes, the PFA can fluoresce, so if you are doing FISH this may be a problem.  For standard ISH, presence of PFA may alter the hybridisation temperature, and/or make the embryos hard to permeabilise.

thanks a lot!