Multicolor Immunohistochemistry for Immune Profiling

For multiple fluorescence immunohistochemistry, Liu et al. can be referred to (26 (link)), and a four-color multiple fluorescence immunohistochemistry staining kit (Absin, Shanghai, China) was used. Briefly, the slices were processed for heat-mediated antigen retrieval by using citric acid buffer. The sections were blocked with goat serum, and then primary antibody was added and incubated overnight at 4°C. On the next day, the sections were washed with TBST and incubated with a second antibody for 15 min, then washed with TBST, and incubated with tyramine signal amplification (TSA) monochrome fluorescent dye for 10 min, after which the above-mentioned steps were repeated. Finally, 4′6-diamino-2-phenylindole (DAPI) was used for nuclear staining. All images were acquired by using Nikon A1 plus a laser confocal microscope. The primary antibodies used for immunofluorescence were anti-CTLA4 antibody (CAL49; 1:500, Abcam), anti-CD4 antibody (EPR6855; 1:500, Abcam), anti-CD4 antibody (EPR19514; 1:1,000, Abcam), anti-CD8 alpha antibody (EPR21769; 1:2,000, Abcam), and anti-CD8 alpha antibody (EPR22483-288; 1:1,000, Abcam).