Vectra automated multispectral microscope

Multiplexed immunofluorescent staining and multispectral image analysis were performed as previously described [42 (link)]. In brief, 4‐mm‐thick formalin‐fixed paraffin‐embedded slides were deparaffinized in a Leica auto‐stainer (Leica Biosystems, Nussloch, Germany), and citrate buffer pH 6.0 (Shanghai Epizyme Biomedical Technology) was used for antigen retrieval. Slides were subsequently blocked with Antibody Diluent (BioGenex Laboratories, Fremont, CA, USA) and incubated with primary antibodies for 30 min at room temperature. Primary antibodies included ARID1A (1:300), CD8 (1:100, C8/1444B, Dako), GB (1:300), PD‐L1 (1:100) and NPM1 (1:200). After washing with TBST, the slides were incubated with horseradish peroxidase‐linked secondary antibodies (Life Technologies, Carlsbad, CA, USA). Afterwards, tyramide‐conjugated fluorophores (Opal, PerkinElmer, Waltham, MA, USA) were added at a 1:50 dilution and incubated for 10 min at room temperature. This process was repeated for all 5 antibodies, and 4’,6‐diamidino‐2’‐phenylindole (Life Technologies) was diluted at 1:500. Finally, slides were cover slipped with VECTASHIELD Hard Mount (Vector Laboratories, Burlingame, CA, USA). Scanning was performed with a Vectra automated multispectral microscope (PerkinElmer), and inForm software (PerkinElmer) was used for analysis.