Antimlh1

IHC staining was conducted on the above sections according to the standardized procedures. Sections were retrieved by EDTA. The primary antibodies used were as follows: antiGBP2 (1:3000 dilution, Cat. 11854-1-AP, ProteinTech), antiPD-L1 (Ready-to-use, Cat. GT2280, GeneTech), antiPD-1 (Ready-to-use, Cat. GT2281, GeneTech), antiCD8 (Ready-to-use, Cat. GT2112, GeneTech), antiMLH1 (Ready-to-use, Cat. GT2304, GeneTech), antiMSH2 (Ready-to-use, Cat. GT2310, GeneTech), antiMSH6 (Ready-to-use, Cat. GT2195, GeneTech), and antiPMS2 (Ready-to-use, Cat. GT2149, GeneTech). Staining was visualized with DAB and hematoxylin counterstain, and stained sections were captured using Aperio Digital Pathology Slide Scanners. For semi-quantitative analysis, the stained sections were independently evaluated by two pathologists. GBP2 and PD-L1 were assessed by according to the evaluation standard on a 12-point scale by calculating the immunoreactivity score (IRS) [31 (link)]. CD8 and PD-1 were assessed by estimating the percentage of cells with strong intensity of membrane staining in the stromal cells.

IHC staining was conducted on the above TMAs and tissue slides. The primary antibodies used in the research were as follows: anti-SECTM1 (1:100 dilution, Cat. 60281-1-Ig, ProteinTech, Wuhan, China), anti-CD8 (Ready-to-use, Cat. PA067, Abcarta, Suzhou, China), anti-PD-L1 (Ready-to-use, Cat. GT2280, GeneTech, Shanghai, China), anti-MSH2 (Ready-to-use, Cat. GT2310, GeneTech, Shanghai, China), anti-MSH6 (Ready-to-use, Cat. GT2195, GeneTech, Shanghai, China), anti-MLH1 (Ready-to-use, Cat. GT2304, GeneTech, Shanghai, China), and anti-PMS2 (Ready-to-use, Cat. GT2149, GeneTech, Shanghai, China). Antibody staining was visualized with DAB and hematoxylin counterstain. All stained sections were independently evaluated by two independent pathologists. For semi-quantitative evaluation of SECTM1 and PD-L1 staining, the H-score criterion was used. In addition, tumors were demarcated into three phenotypes based on the spatial distribution of CD8⁺ T cells, including the inflamed, the excluded, and the deserted subtypes.⁴² (link)^,43 (link) The inflamed subtype is considered to be immuno-hot, and excluded and deserted subtypes are considered to be immuno-cold.⁴⁴ (link)