[Histonet] Trouble with IHC

[Lewis, Patrick]

Hi everyone

I am having some trouble with my IHC on frozen OCT sections.

The antibody/secondary/AEC works great on my Epitope retrieved paraffin sections.

However,

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.

Thanks

Patrick.

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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