I am working on formalin-fixed paraffin-embedded human brain tissue (7um thick), and have been testing out various neuronal antibodies as well as optimising the immunofluorescence protocol itself (such as the antigen retrieval step) but with no success. Has anyone had any luck with the Millipore NeuN raised in mouse, or the HuC/D? Or any other neuronal antibody? (I'm double staining with TFAM (mitochondrial DNA marker raised in rabbit)
You didn't say anething about your protocol - Could it be that your tissue is ovverfixed (!) by formalin?? If so any antibody will not penetrate to the epitope
Hi Elisabeth,
We use the millipore Neun Antibody (MAB377) on FFPE human tissue with great success. After deparraffinisation with xylene and ethanol we currently just add the primary with no retrieval steps at 1:200 followed by biotinylation. It will also work with methanol treatment (-20C for 15 minutes) or 20 minutes 100C in citrate buffer if you need to combine with other antibodies. We also use SMI-32 from biolegends (needs citrate and methanol), chicken neurofilament from abcam (citrate and methanol), rabbit MAP2 millipore (citrate and methanol), mouse MAP2 millipore (citrate and methanol).
Hope it works!
Rachel
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