Immunohistochemical analysis for LAG-3, CD3, GZMB, PD-L1, and PD-1 expression

Pathologically identified, formalin fixed, paraffin-embedded NPC specimens from patients who were biopsied at SYSUCC were retrospectively tested. Archived hematoxylin–eosin staining sections were assessed by two independent pathologists. Immunohistochemical (IHC) staining for LAG-3, CD3, GZMB, PD-L1, and PD-1 expression was conducted using sections obtained from the formalin-fixed diagnostic specimens. Briefly, 4-µm sections were deparaffinized in xylene, rehydrated, and then treated with a citrate antigen restore buffer (pH 9.0) to expose the antigen in the sections. After processing following the conventional steps, the slides were incubated overnight at 4 °C with primary antibodies against LAG-3 (1:200, ab101500, Abcam, Cambridge, MA), CD3 (1:200, ab16669, Abcam, Cambridge, MA), GZMB (1:100, ab255598, Abcam, Cambridge, MA), PD-L1 (M365329, Dako, Carpenteria, CA), and PD-1 (1:50, 315M, Cell Marque, Rocklin, CA). After washing them three times with a phosphate-buffered saline (PBS), 5  min per wash, the sections were sequentially incubated with a Horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (PV6000, ZSGB-BIO, Beijing, China). An evaluation was performed using 3, 3′-Diaminobenzidine (DAB) substrate kits (ZLI-9017, ZSGB-BIO, Beijing, China). The sections were stained with hematoxylin for 4 min and counterstained with bluing reagent for 4 min. The slides were washed and then dehydrated in 70% to 100% alcohol baths followed by xylene baths before coverslip application.