Tissue Immunostaining for Imaging Mass Cytometry

Place a glass slide with a fixed tissue sample into an antigen retrieval buffer and heat it.

The elevated temperature and the buffer's alkaline pH break the fixation-induced protein crosslinks, exposing antigen epitopes.

Cool the slide to prevent heat-induced tissue damage, then rinse to remove excess buffer.

Apply a hydrophobic barrier and place the slide in a humidity chamber.

Add a permeabilization-blocking buffer. The buffer's detergent permeabilizes cellular membranes, and blocking proteins mask non-specific binding sites to reduce background staining.

Remove excess buffer, add a metal-conjugated antibody cocktail, and incubate to allow antibody binding to specific antigens.

Wash the slide, removing unbound antibodies.

Add a metal-conjugated nuclear stain that enters the cells and binds to DNA.

Rinse off excess stain and dry the slide.

Using an optical scanner, obtain high-resolution images of the tissue's structural features, which aids in interpreting data from imaging mass cytometry analysis of the stained sample.