Quantifying Tumor-Infiltrating T Cell Subsets

To confirm the co-expression of CD36 and CD8 on tumor infiltrating T lymphocytes, dual immunofluorescence was performed first, and the infiltration of other immune cells was explored by immunohistochemistry later. Tissue microarray construction and the IHC protocol have been described previously [12 (link)]. The details of the antibodies used in IHC assay are provided in Supplementary Table 3. IHC sections were scanned by Olympus CDD camera, Nikon eclipse Ti-S microscope (200×magnification) and NIS-Elements F3.2 software. Two experienced pathologists, blinded to the follow-up information, counted the number of positive staining cells at 200× magnification, and the average number was used as the final data. For IF staining, the slides were incubated with specific antibodies at 4 °C overnight. Then, samples were incubated with species-appropriate rabbit/mouse secondary antibodies coupled to Alexa Fluor dyes (488 (ab185033),594 (ab203419)) for 1 h at room temperature. DAPI (ab285390) containing anti-fluorescence quenching was used to mount cover slips, and the sections were analyzed through Leica DMi8 microsystems.