[Histonet] Trouble with IHC
patrick.lewis <@t> seattlechildrens.org
Fri Nov 22 15:40:33 CST 2013
I am having some trouble with my IHC on frozen OCT sections.
The antibody/secondary/AEC works great on my Epitope retrieved paraffin sections.
When I try a similar IHC with frozen OCT blocks. Different samples however, but still should be positive.
I get lots of nonspecific background staining and no or impossible to tell specific staining when I run them with no epitope retrieval.
If I try an epitope retrieval on the OCT sections, I get no background but also no specific staining.
My OCT sections are fixed for 30 minutes in 4% p-formaldehyde. Otherwise they are treated pretty much the same way as my paraffin sections (-xylene/etoh of course)
Does anyone have a general IHC protocol for frozen sections with a monoclonal mouse and/or polyclonal rabbit primary antibody with a HRP labeled secondary antibody using an AEC substrate? I want to se if my protocol is off the mark at any step that might cause issues.
Also, I was cutting my OCT sections at 5 um. The slides look a bit holey and chewed up. (I didn't embed these OCT blocks). I am thinking of cutting 8-10 uM sections next time to put a little more tissue on the slide so that they are a little more robust for IHC.
This is my initial attempts to IHC these OCT blocks, so I think a lot of optimization may need to be done, but I was surprised that it would work very well for the paraffin and not at all for the Frozen OCT.
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