Cal49

Total protein was extracted from both cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Abcam, UK). Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). An equal amount of protein from both cells was separated using 12% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane. The blotted membranes were blocked with Tris-buffered saline containing 5% non-fat milk to avoid nonspecific binding sites before the addition of primary antibodies i.e., anti-PD1 (EPR20665, 1:1000, Abcam, UK), anti-CTLA4 (CAL49, 1:1000, Abcam, UK), and anti-beta-actin (EPR21241, 1:5000, Abcam, UK). Beta-actin served as an internal control. After 24 h of incubation at 4 °C, the membrane was incubated again for 1 h with secondary antibody i.e., Immunoglobulin G (IgG) (1:100,000, Abcam, UK) at room temperature. Finally, the proteins were detected using Enhanced Chemiluminescence (ECL) Substrate Kit (Abcam, UK).

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).