I plan to do immunofluorescence staining of microtome sections of paraffin-embedded metastatic tumor tissue with two or more than two antibodies with different emission spectra. Should I follow standard IHC protocol and probe those sections with primary followed by conjugated secondary antibodies? Will this show proper signals of the antibodies and their colocalizations?
Yes, the standard protocol should be fine.
If you get any problems with imaging, drop a line or in private message.
The procedure is the same. After removing paraffin from your slides by xylene, ethanol steps, you can go ahead and add your primary and secondary antibodies and so on. However, which primary to start with, whether to mix primaries and etc depends on the type of antibodies you are going to use and their expected localizations.
Yeah, FFPE sections should be stained like frozen sections for fluorescent double staining.
After deparafinization, Antigen retrieval is also an option before starting the blocking for the staining.
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