Samples were fixed and processed as described in [19 (link)]. The following primary antibodies were used: mouse monoclonal anti-CD3 (clone SP7, 1:70, Leica, #565-LCE) and anti-FOXP3 (1:200, Abcam, ab22510), rat monoclonal anti-Ly6G (clone 1A8, 1:400, Cederlane, #ABF118UD), rabbit polyclonal anti-IBA1 (1:300, Wako, #019-19741), and mouse monoclonal anti-TfR (1:800 #M36008 Invitrogen). Detection was done using EnVision+ System-HRP Labeled Polymer anti-mouse or anti-rabbit (Dako) or using Rat-on-Mouse HRP-Polymer (Biocare Medical) followed by DAB as chromogen. Hematoxylin counterstaining was performed on all sections.
Positive cells count was carried out with Aperio Imagescope on digitalized sections acquired using the Aperio CS2 digital scanner and ScanScope software. The analysis was perform using IHC nuclear Image Analysis and Positive Pixel Count v9 9.0 Algorithm (Imagescope, Leica Biosystem). The whole tumor area was considered for the analysis and necrotic areas were excluded.
Positive cells count was carried out with Aperio Imagescope on digitalized sections acquired using the Aperio CS2 digital scanner and ScanScope software. The analysis was perform using IHC nuclear Image Analysis and Positive Pixel Count v9 9.0 Algorithm (Imagescope, Leica Biosystem). The whole tumor area was considered for the analysis and necrotic areas were excluded.