Immunohistochemical Analysis of Tumor Necrosis

The selected samples of tumor tissues from a xenograft model were fixed, embedded, and stained with 4% paraformaldehyde, paraffin, and H&E staining at a certain temperature to examine tumor necrosis. The remainder slides of tumor tissues were blocked with 3% BSA and incubated with primary antibodies Caspase 3 (1:200) or Ki67 (1:200) for 30 min. Then, the sections were incubated with HRP-conjugated secondary antibodies for 50 min, colored with diaminobenzidine substrate kit (DAKO, K5007), counterstained with Harris hematoxylin for 3 min, and then observed by microscopy. The scores for Caspase 3 and Ki67 immunohistochemistry were calculated according to a previously reported method^{51 (link)}.