Horg c110pt 01

Multiple immunofluorescence staining was performed as previously described (39 (link), 40 (link)). The primary Abs were CD8 (Mouse, 1:3000, Proteintech), CD68 (Rabbit, 1:3000, AiFang biological), CD93 (Rabbit, 1:200, Thermo Fisher), and CD163 (Rabbit, 1:3000, Proteintech). PV6001 (horseradish peroxidase-conjugated secondary antibody, ZSGB-BIO, China) was the secondary antibody, and the tyramide signal was amplified to TSA [FITC-TSA, CY3-TSA, 594-TSA, and 647-TSA (Servicebio, China)]. Image analysis and positive cell quantification were performed by Caseviewer (C.V 2.3, C.V 2.0) and Pannoramic viewer (P.V 1.15.3). Negative controls excluded the primary Ab. We obtained the tissue microarray (HOrg-C110PT-01) from the Outdo Biotech company (Shanghai, China), and the ethics were approved.

Human multiple organ tissue arrays (Cat# HOrgC110PT01 and HOrgC120PG05, Shanghai Outdo Biotech Co., Ltd., Shanghai, China) were used to test the protein expression of PNPO by immunohistochemistry. The study of the human subject was approved by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. A total of 230 paraffin-embedded tissue samples were collected, and of these, 2 samples were either invalid or missing. All the other 228 samples were valid, including 121 malignant tumor tissues, 89 adjacent tissues of cancer, and 18 normal tissues (Supplementary Table S1). The anti-PNPO antibody (1:2000 dilution, #15552-1-AP) was obtained from ProteinTech Group (Chicago, IL, United States).