The levels of CD4, CD8, FOXP3, and CD163 were evaluated in the immune cells located in the invasive tumor area [15 (link)]. CD8 and CD163 were demonstrated by staining methods using antibodies to CD8 (Cell Marque, 108R-14) and CD163 (Biocare Medical, ACR353AK) cells. FOXP3 and CD4 cells were evaluated by the double-staining method using antibodies to CD4 (Biocare Medical, ACI3148) and FOXP3 (Genetex, GTX107737) cells. The MACH 2 Double Stain 2 (Biocare Medical) was utilized for the incubation process. FOXP3 and CD4 cells were stained with Vulcan Fast Red (Biocare Medical) [17 (link)].
Immunohistochemical staining of PD-L1 was performed using a mouse monoclonal primary anti-PD-L1 antibody (clone 22C3; Dako; Agilent Technologies, Inc.). Subsequently, the slides were incubated with Novolink Polymer Detection System (Leica Microsystems) as a secondary antibody (Novocastra). The combined positive score (CPS) was used for evaluating immunohistochemical expression of PD-L1 [19 (link)].
Immunohistochemical staining of PD-L1 was performed using a mouse monoclonal primary anti-PD-L1 antibody (clone 22C3; Dako; Agilent Technologies, Inc.). Subsequently, the slides were incubated with Novolink Polymer Detection System (Leica Microsystems) as a secondary antibody (Novocastra). The combined positive score (CPS) was used for evaluating immunohistochemical expression of PD-L1 [19 (link)].
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