I am having trouble with Nanog IHC in ESCs and iPSCs. In all of the cell lines that I am characterizing, I have measured mRNA expression of Oct4, Sox2, and Nanog, and I have successfully stained Oct4 and Sox2. However, I cannot stain Nanog. I tried multiple protocols (using BSA and/or FBS as blocking reagent) and multiple primary/secondary antibodies (successfully used by other labs, secondary antibody works for Oct and Sox2). Has anyone else had this problem before?
Hi Hayden,
If you are staining cultured cell lines and you successfully stained for Oct4 and Sox2, your staining protocol should be just fine (as you can stain for transcription factors), the problem could be either in the antibodies or the imaging setup, if you have rolled out the antibodies problem, and the imaging setup, it could be actually that Nanog protein expression is not detected in your cells. You might test your Nanog staining on positive control.
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