[Histonet] Re: Question on frozen section IHC and tissue adhesion

[Teri Johnson]

Hi Christina,

I agree with Rene Buesa that ideally you should have added a cryoprotection step prior to freezing. If you snap freeze quickly enough though, you might have gotten away with no freezing artifact. An H&E will tell you that. If it looks like a sponge, or like frost patterns, you might have a specimen that is useless. If not, proceed.

You might try a couple things.

First, cut as thin as you can. 4 microns if you can get away with it. Air dry at least one hour after sectioning, preferably overnight. If you use peroxidase block, dilute your solution to 0.3% and increase your time to 30 minutes. This provides a more gentle approach to endogenous peroxidase blocking. If you can keep the sections on by doing the first two things listed, you can probably use the Alk Phos detection method and not have to worry about using H2O2.

What is the difference in your fluorescence and your chromogen technique besides the H2O2? Fewer steps and washes? Make sure your buffer rinses are done gently, maybe in a coplin jar instead of squirting with a squeeze bottle.

Good luck!

Teri Johnson, HT(ASCP)QIHC
GNF Histology Lab Manager
Genomics Institute of the Novartis Research Foundation
858-332-4752