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. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
