Gb13340

The clinical samples were fixed in 10% neutral buffered formalin for 24 h. Paraffin was used for tissue embedding. Then tissue slides were well prepared and deparaffinized using dimethylbenzene, anhydrous ethanol, 85% ethanol, 75% ethanol, and distilled water orderly. The container with ethylene diamine tetraacetic acid (EDTA) antigen repair buffer (PH 9.0, Servicebio, G1203) for MYD88 or citric acid tissue antigen repair solution (PH 6.0, Servicebio, G1202) for CD68 and CD163 in the microwave oven was used correspondingly to repair the antigen of the slides using median fire to boiling for 8 min, keeping warm for 8 min and median-low fire for 7 min consecutively. Peroxidase was blocked using the 3% H₂O₂ for 25 min. Then we blocked the antigen using goat serum (Servicebio, G5001) for 30 min. We used MYD88 (1:20, CST, 4283S),CD68 (1:200, Servicebio, GB14043) and CD163 (1:600, Servicebio, GB13340) antibody overnight at 4°C to stain the slides, among which the two adjacent slides were stained with CD68 and CD163 separately. Then the slides were incubated with secondary antibodies (1:200, Servicebio, GB23303) for MYD88 and CD163 or secondary antibodies (1:200, Servicebio, GB23301) for CD68 50 min at room temperature. After adding DAB (DAKO, K5007) and hematoxylin (Servicebio, G1004) staining, slides were covered and observed by microscope (Grundium OCUS).

Anti-CD80 monoclonal antibody (66,406-1-Ig, Proteintech), anti-CD163 monoclonal antibody (GB13340, Servicebio), anti-CD8 monoclonal antibody (GB12068, Servicebio), anti-CD31 monoclonal antibody (GB113151, Servicebio), VEGFA Monoclonal antibody (19,003-1-AP, Proteintech), anti-CD47 monoclonal antibody (ab218810, Abcam), carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (C0051, Beyotime), PerCP anti-CD68 (333,813, BioLegend), PE anti-CD11b (101,208, BioLegend), granulocyte–macrophage colony-stimulating factor (GM-CSF) (C003, novoprotein), FITC-labeled anti-CD47 (CC2C6, BioLegend), Human Lymphocyte separation medium (7,111,011, DAKEWE). The fusion protein SIRPα-Fc has been engineered based on the initial extracellular domain of SIRPα and is currently undergoing Phase I/II clinical trial (NCT05140811) [32 (link)]. SIRPα-VEGFR1 is constructed by combining the extracellular domain of SIRPα with that of VEGFR1 (GenBank accession number: MG920788).

Immunohistochemical staining was carried out according to the methods described by Li et al.^{26 (link)}. Specific primary antibodies against claudin-1, occludin, RIP1, and HMGB1 were the same as those used in Western blot. Specific primary antibodies against CD163 (a cell surface marker of macrophage) (#GB13340) and CD11b (a cell surface marker for monocytes) (#GB11058) were purchased from Servicebio. Images were taken using a microscope (Eclipse Ci-L, Nikon, Tokyo, Japan) and analyzed by Image-Pro Plus 6.0 software (Media Cybemetics, MD, USA).