Immunohistochemistry

Tumor tissues were harvested from mice and were cut into 5‐mm‐thick tissue blocks. The tissue blocks were covered with O.C.T. cryo‐embedding media and were frozen with 2‐methyl‐butane cooled by liquid nitrogen. The frozen tissue blocks were sliced into 8‐µm sections and mounted on glass slides (SuperFrost Plus, Thermo Scientific). Sections were fixed in cold acetone for 10 min, dried, incubated in blocking solution (10% FCS, 1% BSA dissolved in 0.01 M phosphate‐buffered saline [PBS], pH 7.2) for 30 min, and then incubated overnight at 4°C with rabbit anti‐cleaved Caspase‐3 (Cell Signaling) dissolved in PBS with 0.5% BSA. The tissue sections were rinsed twice in PBS, and once in PBS with 0.1% Tween, followed by incubation with secondary antibodies (Alexa Fluor 594‐conjugated goat anti‐rabbit) in PBS with 0.5% BSA for 45 min at R.T. Tissue sections were then rinsed twice in PBS and once in PBS with 0.1% Tween, followed by incubation with 0.3 µM DAPI (Invitrogen) in PBS for 15 min at R.T. Finally, the sections were rinsed twice in PBS and mounted with mounting medium. Images were acquired with the Leica SP8 confocal inverted microscope under a 63× objective lens.