H&E staining was performed on the TMA specimens, and the results were assessed by an experienced pathologist. For the immunohistochemistry (IHC) staining, anti-CD8 (Cell Signaling Technology, Danvers, Massachusetts, USA) and anti-FoxP3 (R&D Systems, Tustin, California, USA) monoclonal antibodies were utilized, as previously described.20 21 (link) The average number of tumor-infiltrating immune cells per high-power fields (HPF) were assessed by three independent ×400 HPF.
Immunofluorescence (IF) staining was performed on the TMA specimens of FFPE tissue. In this study, we used anti-myeloperoxidase (MPO) (Abcam, ab208670, Cambridge, UK), anti-CD11b (Abcam, ab133357), and anti-CD206 (Abcam, ab64693) monoclonal antibodies as the primary antibodies at room temperature for 1 hour of incubation. Next, the specimens were incubated for an additional hour with Alexa Fluor 488 (B40953), Alexa Fluor 555 (B40955), and Alexa Fluor 647 (B40958) along with Tyramide Reagent (ThermoFisher Scientific, Waltham, Delaware, USA). As the final step, the specimens were incubated for 20 min with DAPI (Solarbio, #C0060). A fluorescence microscope (Olympus, Tokyo, Japan) was used to visualize the cells.
Immunofluorescence (IF) staining was performed on the TMA specimens of FFPE tissue. In this study, we used anti-myeloperoxidase (MPO) (Abcam, ab208670, Cambridge, UK), anti-CD11b (Abcam, ab133357), and anti-CD206 (Abcam, ab64693) monoclonal antibodies as the primary antibodies at room temperature for 1 hour of incubation. Next, the specimens were incubated for an additional hour with Alexa Fluor 488 (B40953), Alexa Fluor 555 (B40955), and Alexa Fluor 647 (B40958) along with Tyramide Reagent (ThermoFisher Scientific, Waltham, Delaware, USA). As the final step, the specimens were incubated for 20 min with DAPI (Solarbio, #C0060). A fluorescence microscope (Olympus, Tokyo, Japan) was used to visualize the cells.