Epr21769

Paraffin tumor sections were prepared following routine protocols for immunohistochemistry. Sections were stained with anti-CD8α (1:500 dilution; Abcam, EPR21769) and then with the secondary antibody conjugated with Alexa Fluor 568 (1:500 dilution; Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratory). Immunofluorescence-stained slides were visualized with a fluorescent microscope aided by Slidebook software (Olympus). The number of CD8⁺ cells was counted in randomly picked four fields of each slide. The number of CD8⁺ staining was directly counted or quantified using the particle analysis tool of ImageJ software (National Institutes of Health). The percent abundance of CD8⁺ staining was calculated by normalizing CD8⁺ count to the DAPI⁺ count.

To evaluate the changes in immunogenic microenvironment in treated tumors, we collected tumors seven days after the last treatment and incubated paraformaldehyde-fixed tumor tissue sections with primary antibodies (CD8, PD1) for 2 h at 37 °C and then washed them with PBS three times. CD8 (EPR21769, Abcam) and PD1 (EPR20665, Abcam) were purchased from Abcam company. Image-Pro Plus 6.0 was adopted to analyze each, and mean density (IOD/area) was used to analyze protein expression.

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).