Is there any specific reason why you don't do typical X-Gal staining with your tissue? With this method, you get staining by the enzymatic activity and are not dependend on an antibody.

Thank you for your response, the reason is that I want to do double staining to localize the protein of interest with different proteins.

Use both primary antibodies from different species. Make sure antibody is validated for mice tissue and no cross reactivity against other protein isoforms.

Try reducing the amount of primary and secondary antibody.

Increase the blocking time.

thank you for your response, both antibodies are validated in mice tissues.

and i could be helpful to alter blocking and concentrations.

thanks

I had a similar problem for a long time. I was working with Fixed-Frozen sections but now use Paraffin sections and include the Perkin Elmer Tyramide Signal Amplification kit and it has helped. I use CAT# NEL702001KT. Good luck :)

thank you so much for you response, I appreciate it.