Immunohistochemical Analysis of Tumor Tissue

After conventional paraffin embedding, the human and mouse tumor tissues were cut into 4-μm histological sections, dewaxed with xylene, and hydrated with gradient density alcohol [16 (link)]. Antigen retrieval was conducted by boiling the sections for 10 min at 100°C in 10 mmol/L citric acid buffer (pH = 6.0). The sections were incubated with antibodies to E2F1 (1:500, sc-251, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), SESN3 (1:100, H00143686-M02, Abnova, Walnut, CA, USA), and ki-67 (1:1000, sc-23,900, Santa Cruz) at 4°C overnight. Next, the sections were re-probed with HRP-coupled anti-mouse secondary antibody (1:2000, ab205719, Abcam Inc., Cambridge, UK) for 60 min at room temperature. Peroxidase activity was observed with diaminobenzidine tetrahydroxyl chloride solution (Vector Laboratories, Inc., Burlingame, CA, USA), and the sections were counter-stained with hematoxylin. After gradient density alcohol dehydration, xylene dewaxing, neutral resin (Merck Millipore, Darmstadt, Germany) sealing, the staining results were observed under a microscope (Nikon Instruments Inc., Melville, NY, USA).