I am currently optimising a protocol for triple staining of murine wounds. I am using MAC-3 (rat), Arg-1 (mouse) and iNOS (rabbit) to characterise the macrophage population in the wound. As secondary antibodies I am using Goat Anti-rat AlexaFluor 488, Goat Anti-mouse AlexaFluor 555 and Goat Anti-rabbit AlexaFluor 647 and then mounting them with Vectashield. They all work individually but when I try to do triple staining they do not. Could anyone help? I am attaching the protocol for the triple staining as well.

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