Hello!
I'm trying to validate CTLA-4 antibody, but until now the only staining I have is 1:50 with heating and enzymatic pre-treatments. However, the tissue is very damaged in the end, even after many protocol settings (only one pre-treatment, longer pre-treatment, formalin post-fixation). The enzymatic or heating step alone causes no damage, but no staining neither. There is some way to get a staining and conserved tissue morphology or should I change the antibody?
Thanks a lot!
Tissue damage in what sense? Antigen retrieval by microwave method is better compared to pressure cookers. Keep the sections in an incubator under controlled conditions if there is a time lag between antigen retrieval and staining.
Incomplete clearing of paraffin wax from the sections may lead to stain not taking up at times.
Basically look for any deficiencies occurring the entire process of your staining protocol.
Hi Layane
What tissue do you use? Fatty tissues are known to be sensitive to "rigorous" treatments.
Maybe you can use slides with a different coating. (APES, Glutaric aldehydy, Poly-L-Lysin)
With kind regards,
Ronald
Hello Vinay and Ronald,
thank you for the answers. I actually use Leica Bond automated stainer and breast cancer tissue, so I only see the results in the end of the run. The tissue is not detached from the slide, but it is so damaged that it looks like a blur (I can't determine individual epithelial cells).
Thank you very much!
Hi Layane,
If your cells are a blur, you could try changing the ezymatic treatment. (Less enzyme, shorter treatment.) I have never seen this blurring because of a cooking step.
Regards,
Ronald
Hi, I do not have experience working on an automated machine. I guess you could try Ronald's suggestions and see if it works.
Regards.
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