Antibody diluent block

Formalin‐fixed/paraffin‐embedded samples from the patients included in this study were collected from Peking University People's Hospital. All the samples were cut into sections of 4‐μm thickness. The slides were deparaffinized in xylene for 30 min and rehydrated in absolute ethyl alcohol for 5 min (twice), 95% ethyl alcohol for 5 min, 75% ethyl alcohol for 2 min sequentially. Washed the slides with distilled water three times. A microwave‐oven is used for heat‐induced epitope retrieval, and during epitope retrieval the slides were I immersed in boiling EDTA buffer (pH 9.0; ZLI‐9069; Zsbio, Beijing, China) for 15 min. Antibody Diluent/Block (72424205; Perkin‐Elmer, MA, USA) was used for blocking. The antibodies used were anti‐CD4 (abcam, ab133616), anti‐CD8 (abcam, ab237709), anti‐CD19 (abcam, ab134114), anti‐CD57 (abcam, ab220187), anti‐CD11b (abcam, ab52478), anti‐CD31 (abcam, ab76533), anti‐pan cytokeratin antibody (abcam, ab32570) and anti‐PDGFR alpha + PDGFR beta (abcam, ab32570). The antigenic binding sites were visualized using the Opal 7‐Color Manual IHC Kit (Perkin‐Elmer, NEL811001KT) according to the manufacturer's protocol. Multicolour immunohistochemistry data were collected using a Mantra Quantitative Pathology Workstation (Perkin‐Elmer, CLS140089) and analysed by InForm (version 2.2.1).

Biopsy tissue and postoperative surgical tissue samples collected before ICI treatment were processed into paraffin blocks and then cut into 4-μm-thick FFPE sections. Staining of the 4-μm FFPE slides was performed by using the Opal Seven-color IHC Kit (NEL797B001KT; PerkinElmer, Massachusetts, USA). The immune markers evaluated included CD8 (ZA-0508, clone SP16; Zsbio; 1:100), CD68 (ZM-0060, clone KP1; Zsbio; 1:400), CD163 (ZM0428, clone 10D6, Zsbio; 1:200), and PD-L1 (CST13684, clone E1L3N, CST, 1:100). Markers were identified and quantified by mIHC. Briefly, sections were cut from tumor tissue, deparaffinized, rehydrated, and washed in tap water before epitope retrieval/microwave treatment (MWT). Endogenous peroxidase activity was blocked using Antibody Diluent/Block (72424205; PerkinElmer, Massachusetts, USA). Protein blocking was performed using Antibody Diluent/Block. One antigen required one round of labeling, including primary antibody incubation, secondary antibody incubation, and TSA visualization, followed by labeling with the next antibody. Slides were scanned using PerkinElmer Vectra (Vectra 3.0.5; PerkinElmer, Massachusetts, USA). The percentage of positively stained cells among all nucleated cells was counted.