Anti cd163 antibody

Hearts were fixed with 20% formalin (Sakura Finetek Japan Inc.) and embedded in paraffin. After cutting 4 µm each, paraffin sections were stained with anti-CD45 (BD Biosciences, USA), anti-F4/80 antibodies (Bio-Rad, USA), anti-iNOS antibody (abcam, UK), and anti-CD163 antibody (abcam) to evaluate cell infiltration. Elastica van Gieson staining and wheat germ agglutinin staining were performed to examine fibrosis proportion and calculate cell area, respectively. Cross-sectional area (CSA) of myocytes with a centrally located nucleus and overall circular shape was measured in the left ventricular free walls using Image J (NIH, USA)^{27 (link)}.

Sections (5µm thick) of formalin‐fixed, paraffin‐embedded glioma tissues were deparaffinized and rehydrated and then incubated with Tris‐EDTA buffer (pH 9.0) for antigen retrieval. Thereafter, the tissue samples were incubated with primary antibodies for 2 hours at ambient temperature (anti‐IFI30 antibody, 1:10 000 dilution, Invitrogen, Carlsbad, CA, USA; anti‐CD163 antibody, 1:200 dilution, Abcam, Cambridge, UK; anti‐PD‐L2 antibody, 1:200 dilution, Proteintech, Rosemont, IL, USA; and anti‐IL‐10 antibody, 1:200 dilution, Proteintech). Then, the sections were rinsed, incubated with appropriate secondary antibodies (ZSGB‐Bio, Beijing, China), treated with 3,3′‐diaminobenzidine staining solution, and counterstained with Mayer's haematoxylin. The staining results were reviewed independently by two investigators.

Detailed information is presented in Additional file 1. Tumor samples were collected from gastric cancer patients who did not receive any preoperative therapy and patients receiving neoadjuvant anti-PD-1 therapy and curative surgery. All staining was conducted on sections of formalin-fixed paraffin-embedded tumor tissues. Basophils were stained using anti-Pro Major Basic Protein 1 (ProMBP1) antibodies (Biolegend, San Diego, USA, catalog number: 346802). M2 macrophages were assessed using an anti-CD163 antibody (Abcam, Cambridge, UK, catalog number: ab182422). For IHC, the slides were incubated with peroxidase-conjugated secondary antibodies, stained using diaminobenzidine (DAB)-H₂O₂, and counterstained with hematoxylin. For IF, fluorescence-labeled secondary antibodies were used and DAPI was counterstained.

IHC staining was used to validate the immunophenotypes in the AHMU‐PC cohort. CD163 (Anti‐CD163 antibody: Cat. ab182422, Abcam Inc., Cambridge, MA, USA) was chosen as the cell marker for macrophages, while α‐SMA (anti‐α‐SMA antibody: Cat. Ab7817, Abcam Inc., Cambridge, MA, USA) was employed to reflect stromal activation and distinguish the immune‐activated and immune‐suppressed subtypes. The detailed steps of the IHC procedure have been previously reported [23, 24]. α‐SMA is universally expressed in stromal cells, and we used the positively stained region score (0, negative; 1, 1%–10; 2, 11–50%; 3, 51–80%; and 4, >80% positive area) multiplied by the immunostaining intensity score (0, no staining; 1, weak; 2, mild; and 3, strong intensity) to semiquantify the results. For the CD163 staining, we directly used ImageJ software (NIH, Bethesda, USA) to count positively stained cells [25].