Tumor sections (three for each tumor) were stained with hematoxylin-eosin (HE) as described (36 (link)). HE stained tumor tissues were examined with a microscope at 400 × magnification.
Tumor sections (five for each tumor) were immunostained with antibodies for cleaved caspase-3 and CD3 expression. Rabbit anti-mouse cleaved caspase-3 antibody and rabbit anti-mouse CD3ε antibody (1:200 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). The sections were treated with antigen retrieval solution followed by 3% hydrogen peroxide treatment, blocked with 5% goat serum, incubated with primary antibody and with biotin-labeled secondary antibody (goat anti-rabbit; 1:1,000 dilution; Abcam, Cambridge, UK). The stained tissues were visualized by using the DAB chromogen (Abcam) reagent and counterstained with hematoxylin. For quantification of immunohistochemical staining, positive cell percentages were counted in five random 400 × microscopic fields for each tissue section.
Tumor sections (five for each tumor) were immunostained with antibodies for cleaved caspase-3 and CD3 expression. Rabbit anti-mouse cleaved caspase-3 antibody and rabbit anti-mouse CD3ε antibody (1:200 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). The sections were treated with antigen retrieval solution followed by 3% hydrogen peroxide treatment, blocked with 5% goat serum, incubated with primary antibody and with biotin-labeled secondary antibody (goat anti-rabbit; 1:1,000 dilution; Abcam, Cambridge, UK). The stained tissues were visualized by using the DAB chromogen (Abcam) reagent and counterstained with hematoxylin. For quantification of immunohistochemical staining, positive cell percentages were counted in five random 400 × microscopic fields for each tissue section.