Cy3 and fitc conjugated secondary antibodies

The protocols used for IHC and immunofluorescence were performed according to previous studies [22 (link)]. Anti-GBE1 (1:300; Abcam, USA), anti-PD-L1 (1:300; Cell Signaling Technology, USA), CD8 (1:300; Abcam, USA), CCL5 (1:300; BioLegend, USA), CXCL10 (1:300; Ruiyingbio, China) were used as primary antibodies. For IHC, three fields of view per sample were imaged. The intensity of immunostaining was taken into consideration when analyzing the data. The percentage scoring of immunoreactive tumor cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), 3 (dark brown). The intensity of staining was obtained by multiplying the two items into a total score, and the scores ranged from 0 to 9. In immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (BioLegend, California, USA, 1:500) were used to detect the primary antibodies. Nuclear staining was performed with DAPI (1:1000; Roche, USA). The samples were visualized with a fluorescence microscope (Olympus, IX71, Japan).

Immunohistochemistry and immunofluorescence were performed as previously described^{48 (link)}. Anti-GBE1 (1:300; Abcam), anti-Ki-67 (1:300; Abcam), anti-caspase 3 (1:300; Abcam), and anti-HIF1α (1:300; Proteintech, Rosemont, IL, USA) were used as primary antibodies. For immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (1:500; BioLegend, San Diego, CA, USA) were used to detect primary antibodies. The samples were then imaged using a fluorescence microscope (IX71; Olympus, Japan).

The protocols used for immunohistochemistry and immunofluorescence are described elsewhere [18 (link)]. Anti-CCL20, anti-CD326, anti-FOXP3 (1:300; Abcam, USA), anti-P-P65, anti-FOXO1, and anti-CEBPB (1:300; Cell Signaling Technology, USA) were used as primary antibodies. For immunohistochemistry, three fields of view per sample were imaged. The intensity of immunostaining was considered when analyzing the data. The percentage scoring of immunoreactive tumor cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). Staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), and 3 (dark brown). Immunoreactivity scores (IRS) were obtained by multiplying the two items to a total score and ranged from 0 to 9. Protein expression levels were further analyzed by classifying IRS values as low (based on an IRS value ≤5) and as high (based on an IRS value > 5). For immunofluorescence, the sections were treated with 1% Triton X100 in 0.01 M PBS. Cy3- and FITC-conjugated secondary antibodies (1:500; BioLegend, USA) were used to detect the primary antibodies. Nuclear staining was performed with DAPI (11,000; Solarbio, China). The samples were visualized using a fluorescence microscope (Olympus, IX71, Japan).