I am trying to stain for a receptor in feline kidneys (not been documented before), with the eventual aim to quantify it. I have tried a few antibodies with similar unsatisfactory results (pictures attached). Using mouse kidneys as a control, primary Ab is raised in the rabbit.

I am using FFPE samples, citrate antigen retrieval, TBS +/- triton buffer, Rabbit specific HRP/DAB (ABC) Detection IHC Kit from Abcam.

My main questions/concerns:

-Overstaining/non-specific staining in the sample slides (both mouse & cats)

-Staining in the primary Ab only slides (second cat > first cat, former is also of a different darker quality)

-> does this suggest I need to use a lower primary Ab concentration?

-Staining (first cat > mouse) of the secondary Ab only slides

-> do I need to change my blocking steps?

-General iridescent 'drops' across some of the slides (mouse negative control & sample in particular)

-> is this staining or something unrelated e.g. crystals in the buffer?

Thank you for any suggestions, I don't have anyone in my lab with IHC experience to help me troubleshoot!