Cd8 antibody

Multiplexed fluorescence staining was performed as previously described (39 (link) {Song, 2022 #205)}. In brief, 4-μm FFPE TMAs sections were deparaffinized in xylene and then rehydrated in 100, 90, and 70% alcohol successively. Antigen unmasking was performed with a preheated epitope retrieval solution, endogenous peroxidase was inactivated by incubation in 3% H2O2 for 20 min. Next, the sections were pre-incubated with 10% normal goat serum and then incubated overnight with primary antibodies panel: CD4 antibody (CST, 48274, 1:100), CD8 antibody (CST, 55336, 1:300) and CXCR6 antibody (abcam, ab273116,1:1000). Next, TMA sections were incubated with the corresponding HRP-conjugated goat anti-rabbit second antibodies (ZSBIO, CA) for 10-30 mins at room temperature. The antigenic binding sites were visualized using the OPAL dye: Opal -650 (AKOYA),Opal −520 (AKOYA), Opal- 570 (AKOYA) for each antibody, respectively. Then, TMA sections were counterstained with DAPI (Sigma) for 3-5 mins. Similar to the data analysis of immunohistochemistry, Multiplexed fluorescence staining images were analyzed and quantified by HALO software (Indica Labs, Corrales, NM, USA) as well. For the convenience of downstream comparisons, the CD4⁺CXCR6⁺, CD4⁺CXCR6^-, CD8⁺CXCR6⁺ and CD4⁺CXCR6^- were analyzed, respectively.

For IHC staining, paraffin-embedded tissue slides were deparaffinized by xylene and hydrated in an ethanol gradient. Then, antigen retrieval was carried out in citrate buffer (pH 6.0) at high-temperature. After blocking with 10% normal goat serum, the tissue sections were incubated respectively with a primary CD163, FoxP3 and CD8 antibodies (1:400, Abcam, USA) overnight at 4°C. Subsequently the slides were treated with the ChemMate Dako EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse kit (DakoCytomation, Glostrup, Denmark) and counterstained with hematoxylin. The immunostaining signals were observed using Nikon microscope.

Immunophenotyping of the splenocytes isolated from the treated and untreated groups were performed using CD4, CD3 and CD8 antibodies (Abcam, USA) via flow cytometry (BD, USA). The differences between the control or treated group and untreated group were determined by one-way ANOVA.