We are staining 16um sections of mouse penis with a PGP9.5 (pan-neuronal) AB. Without paraffin the staining was very nice but sections dissociated. Now we embed in paraffin but the staining is much weaker. After paraffin removal we boil sections in citrate buffer, block with BSA+Triton and put on the primary AB in PBS-X.
Hi Johanna,
During antigen retrieval process, you used boiling. Sometime boiling the sample may damage the antigen of interest and produce poor signal. You may try mild antigen retrieval processes. Here is a link for different antigen retrieval processes (http://www.ihcworld.com/epitope_retrieval.htm). You may try low temperature heating process so that the antigen is less damaged.
In our lab we do a lot of OCT embedded cry frozen section (12um) and the staining is very nice for different antibody. You may think about frozen section as well. The blocking and permeabilzation process is same as you mentioned. Thank you!
Dear Johanna.
The most common reason why an AB doesn´t work on paraffin sections is antigen masking. Then antigen retrieval could fix the problem. You could try the methods Md Riaj Mahamud suggested by posting the IHC-World link.
The setup with different AG-retrieval methods I usually use is:
- No AG-retrieval
- Boiling in NaCitrat-buffer pH 6 (this didn´t work for you)
- Boiling in Tris/EDTA-buffer pH 9
- 8 min/37°C Proteinase XXIV in 20mM Tris pH 9
You can also try different primary and/or secondary AB concentrations. And don´t forget to include a control without primary, just to be sure that the staining you see is not background.
If none of this works, probably the AB you are using is not suitable for paraffin sections. You could try a new one:
https://www.biocompare.com/Antibodies/
Hope this helps.
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