H&E staining and Immunohistochemistry were performed as previously described (15 (link), 16 (link)). Briefly, tumor specimens were first fixed with 10% formalin for 24 h and 70% ethanol for 12 h prior to embedding in paraffin and sectioning. Tissue sections were then deparaffinized in xylene and rehydrated in graded ethanol.
For H&E staining, after staining with hematoxylin for 5 minutes, sections were stained with eosin solution for 30 seconds. Next, sections were dehydrated and mounted using neutral gum.
For IHC, heat-induced antigen unmasking was performed in a 10mM citrate buffer and then blocked with 1% BSA for 1 h at room temperature. Sections were incubated with primary antibodies at 4°C overnight at an ideal dilution in a humidified chamber. On the next day, sections were incubated with secondary antibodies for 30 minutes at room temperature (Zsbio, pv8000), followed by detection using the DAB detection kit (OriGene Technologies, ZLI-9017). The following primary antibodies were used: anti-Ki67 (Abcam, ab15580; 1:100), anti-F4/80 (Cst,70076; 1:200), anti-CD20 (Abcam, ab64088; 1:100), anti-CD4(Abcam, ab183685; 1:100), anti-CD8 (Abcam, ab228965; 1:100), anti-Collagen type IV (Affinity, af0510; 1:100), anti-CK7 (PTG, 17514-1-Ap; 1:100), anti-CD34 (Abcam, ab81289; 1:100), and anti-Ly6G (Invitrogen, 2335909; 1:100).
For H&E staining, after staining with hematoxylin for 5 minutes, sections were stained with eosin solution for 30 seconds. Next, sections were dehydrated and mounted using neutral gum.
For IHC, heat-induced antigen unmasking was performed in a 10mM citrate buffer and then blocked with 1% BSA for 1 h at room temperature. Sections were incubated with primary antibodies at 4°C overnight at an ideal dilution in a humidified chamber. On the next day, sections were incubated with secondary antibodies for 30 minutes at room temperature (Zsbio, pv8000), followed by detection using the DAB detection kit (OriGene Technologies, ZLI-9017). The following primary antibodies were used: anti-Ki67 (Abcam, ab15580; 1:100), anti-F4/80 (Cst,70076; 1:200), anti-CD20 (Abcam, ab64088; 1:100), anti-CD4(Abcam, ab183685; 1:100), anti-CD8 (Abcam, ab228965; 1:100), anti-Collagen type IV (Affinity, af0510; 1:100), anti-CK7 (PTG, 17514-1-Ap; 1:100), anti-CD34 (Abcam, ab81289; 1:100), and anti-Ly6G (Invitrogen, 2335909; 1:100).