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.

Begin with a five-micrometer pre-treated tissue section mounted over an immunostaining-compatible slide. Place the slides in a slide mailer.

For heat-induced epitope retrieval or HIER, add HIER buffer in the slide mailer until the tissue is completely immersed. Place the slide mailer in a decloaking chamber, and start the heating program.

After the program ends, place the slide mailer in a tray filled with cold water, and allow it to cool down. Rinse the slides with distilled water several times by gradually increasing the volume.

Remove the slides from the slide mailer, and wipe off the excess buffer. To prevent loss of blocking buffer and antibodies, encircle the tissue using a hydrophobic barrier pen.      

Incubate the slides in a customized humidity chamber for one hour. For non-specific blocking, add blocking buffer to the slides in the humidity chamber. After the incubation period, remove the excess blocking buffer, and let the remaining buffer air-dry at room temperature.

Carefully add the antibody solution over the tissue and incubate overnight in a humidity chamber at four degrees Celsius.

Following incubation, wash the slide with Triton-containing PBS for 5 minutes. Finally, rinse the slide twice with PBS for 5 minutes each. Now, treat the tissue with the intercalator dye and incubate it in the humidity chamber at room temperature for thirty minutes.

After incubation, wash the slide with HPLC-grade water for five minutes, and dry it in a desiccator for thirty minutes.

Once the slide is dry, scan using a flatbed document scanner at a resolution not less than 1200 dots per inch.