I have encountered issues performing fluorescent IHC on rat spinal cord cross-section to detect p-NR1 (SER 896). The antibody used in "ABN88" from millipore. While I do see positive signal compared to my negative control (without the primary antibody, only secondary FITC), the staining pattern is not like any reported in the literature. Others report peri-somal staining which is to be expected, as this is a subunit of the NMDA receptor. However, I see intense nuclear staining which is very unanticipated. In addition, I've found no convincing signs of the peri-somal staining that others see.
Has anyone else encountered these issues with this marker, or perhaps had better luck with it? I'm currently considering citrate buffer antigen retrieval in case my problem has to do with epitope exposure.
Thanks!
Hello Khalid.
If you take a look at the IHC-picture on the millipore website they state, that the staining worked on paraffin-embedded tissue using heat-induced epitope retrieval in citrate buffer pH 6.0. Immunostaining was performed using a 1:25 dilution of the ABN88-ab.
http://www.merckmillipore.com/DE/en/product/Anti-phospho-NR1-%28Ser896%29-Antibody,MM_NF-ABN88#relations
Maybe you should try this!
Greetings
what is the temperature preferring for the antigen retrieval step?
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