I did IHC using fluorescent labeled secondary antibodies to detect astrocytes with GFAP marker. My sample was fixed frozen mouse brain tissue streatum region. I saw fluorescence in my sample treated without the primary antibody. I noticed that this fluorescence was exactly overlapped the DAPI staining. I used alexafluor488 secondary antibody and mounting media with DAPI. I attached the picture with my staining. My secondary antibodies were at a 1:1000 dilution.
I believe it is the microscope parameter. The image seems have taken from the same channel, if you merge it you can see both colors come from the same spot. Make sure your parameters are right: check DAPI at 405 and Alexa at 488
This appears to be a possible case of natural autoflorescence. Would there be a problem going to a red flouresence marker that was further from an FITC range? The effects of endogenous autoflourescence would be minimized.
Just for thoroughness, could be good to take a look at a unstained sample using the same imaging parameters as well.
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