Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Take a renal cell carcinoma tissue cryosection exhibiting infiltrating CD8+ T cells in its tumor microenvironment.

Treat it with acetone to fix and permeabilize the tissue. Wash to remove excess acetone.

Add avidin to saturate endogenous tissue biotin. Next, add biotin to block immobilized avidin-binding sites, minimizing non-specific interactions.

Apply serum containing immunoglobulins to block immune cell Fc receptors, preventing non-specific antibody binding.

Add a primary antibody cocktail targeting CD8, a cytotoxic T cell marker, and PD-1 and Tim-3, inhibitory receptors expressed by CD8+ T cell subpopulations resistant to immunotherapy.

Introduce distinct fluorophore-labeled secondary antibodies targeting anti-CD8 and anti-Tim-3 antibodies.

Add biotinylated secondary antibodies targeting anti-PD-1 antibodies and a different fluorophore-labeled streptavidin binding to biotin.

Apply a dye-containing mounting medium to stain cell nuclei and place a coverslip.

Acquire multispectral fluorescence images to identify the tumor-infiltrating CD8+ T cells and the immunotherapy-resistant subpopulations expressing PD-1, Tim-3, or both.