I have to stain tissue (Fixed rat brain, free-floating) with two different primary antibodies (one is a mouse monoclonal and the other is a goat polyclonal). My secondary fluorescent conjugated antibodies are goat anti-mouse Alexa 488 &  donkey anti-goat Alexa 568. 

I was told that I could stain the tissue by incubating with the antibodies sequentially starting with the goat antibody (for 24 hrs) first followed by its secondary (for 3 hrs) then incubating with the mouse antibody (for 24 hrs) followed by its secondary (for 3 hrs). However, the procedure does not seem to work. I only see staining for the goat antibody and not for the mouse antibody. I know that both antibodies work individually. 

Does anyone have suggestions? Is it the secondary antibodies that are causing the problems. 



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