A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

Take a fixed and permeabilized adrenal section comprising a cortex and a medulla. Treat it with a boiling acidic buffer, breaking the fixation-induced crosslinks for antigen unmasking.

Block the non-specific binding sites and introduce primary antibodies binding to cortex-specific markers.

Add biotinylated secondary antibodies targeting the primary antibodies and streptavidin-conjugated peroxidases binding to biotin.

Introduce a fluorophore-tagged tyramide and hydrogen peroxide.

The immobilized peroxidases utilize hydrogen peroxide to activate tyramide, which binds to nearby proteins, labeling the cortex.

Next, treat the section with the boiling buffer to strip the bound antibodies.

Introduce the same host species-generated primary antibodies targeting medulla-specific markers and biotinylated secondary antibodies.

Introduce streptavidin-conjugated peroxidases and a different fluorophore-conjugated tyramide to label the medulla.

Stripping the cortex-specific primary antibodies inhibits the reintroduced secondary antibody binding them, preventing cross-reactivity.

Apply a nuclear stain and perform imaging to visualize the distinctly stained cortex and medulla, confirming no antibody cross-reactivity.