The changes in infiltration levels of 21 immune cells were evaluated through the CIBERSORT algorithm, which quantifies the cell composition of sample tissue based on the gene expression profiles (21 (link)).
To verify the immunogenic effects of NOS2, we performed immunofluorescence detections on two pairs of clinical samples with different NOS2 protein levels. Immunofluorescence was conducted as previously described (22 (link)). Briefly, 4-mm tissue sections of HB clinical samples were prepared, and dewaxed with xylene and anhydrous ethanol. Then, the tissue sections underwent antigen repair, fixation, and nonspecific blocking. Next, the sections were incubated with first and secondary antibodies (NOS2, ab178945; CD8, ab33786; CD163, ab87099; Abcam, Cambridge, UK) in conditions protected from light. The slides were then sealed with anti-fluorescence quenching sealing tablets. CD8 and CD163 were stained with the Cy3-labeled goat anti-mice IgG (H&L) antibody (P0193; Beyotime, Shanghai, China). NOS2 was stained with the FITC-labeled goat anti-rabbit IgG (H&L) antibody (P0186; Beyotime). Nuclei were stained with Hoechst reagent (C0003; Beyotime). Subsequently, the slides were analyzed with an automatic fluorescent microscope using a 40× objective lens (BX53; Olympus, Tokyo, Japan).
To verify the immunogenic effects of NOS2, we performed immunofluorescence detections on two pairs of clinical samples with different NOS2 protein levels. Immunofluorescence was conducted as previously described (22 (link)). Briefly, 4-mm tissue sections of HB clinical samples were prepared, and dewaxed with xylene and anhydrous ethanol. Then, the tissue sections underwent antigen repair, fixation, and nonspecific blocking. Next, the sections were incubated with first and secondary antibodies (NOS2, ab178945; CD8, ab33786; CD163, ab87099; Abcam, Cambridge, UK) in conditions protected from light. The slides were then sealed with anti-fluorescence quenching sealing tablets. CD8 and CD163 were stained with the Cy3-labeled goat anti-mice IgG (H&L) antibody (P0193; Beyotime, Shanghai, China). NOS2 was stained with the FITC-labeled goat anti-rabbit IgG (H&L) antibody (P0186; Beyotime). Nuclei were stained with Hoechst reagent (C0003; Beyotime). Subsequently, the slides were analyzed with an automatic fluorescent microscope using a 40× objective lens (BX53; Olympus, Tokyo, Japan).