Hello,
I am trying to mark cells undergoing proliferation in the brain of adult zebrafish (Danio rerio). I did immunohistochemistry on sections using the Anti-PCNA antibody [PC10] (ab29) with antigen retrieval but did not get any positive staining. Did anybody try this antibody and if so under which conditions? Indicating the secondary antibody would also be very useful ( I used Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488).
Thank you so much for your help
Zebrafish proteins usually have quite a poor immunoreactivity even now with a wide array of monoclonal antibodies with many predicted to work with zebrafish achieving a good signal/good specificity standing is zebrafish for many protein targets is a challenging task. I would suggest doing Edu/BrdU incorporation as it chemical based you will see all proliferating cells in the brain.
Also, zebrafish a fairly autofluorescent at 488 nm so in your future immunostaining consider switching further into the red spectrum Cy3 or Cy5/Alexa647 is preferable. Note though that Cy5 is an infrared emission dye and can only be observed by the microscope camera.
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