[Histonet] RE: Trouble with IHC
TJohnson <@t> gnf.org
Mon Nov 25 10:33:34 CST 2013
It sounds as though you have a lot going on. It's important to gauge whether your frozen sections are moth-eaten due to trimming/facing the tissue too aggressively and tearing chunks of tissue out, if it's freezing artifact, or if your sample is too cold and you are getting chatter artifact.
As for your IHC, it could be that your antibody is optimized for formalin fixed, paraffin embedded samples. You can fixing the tissues longer, you can even post fix again after sectioning and before staining and see if that helps. Or it could be that you need to use some heat induced retrieval for it to recognize the protein, and we have successfully used retrieval at 60 degrees C on fixed frozen sections. We avoid the high temp (>= 95 degrees C) as it could cause more damage to the tissue and epitopes.
You may also think about post-fixing slides with a formal-alcohol, zinc formalin, or alcohol-acetone and see if any of those work for your sample.
Do you have an antibody that is known to work in frozen section? If so, use this to optimize your detection system. Once that is optimized, it is easier to then optimize staining conditions with unknown antibodies.
Which antibody are you trying to stain and what tissue are you using?
Genomics Institute for
San Diego, CA
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