Ab279653

The expression of KI67 was detected by Immunohistochemistry. The sections were isolated from tumor tissues and then treated with conventional deparaffinization, rehydration, and blocking of endogenous peroxidase activity for 15 min. Sections were pretreated for the purpose of antigen retrieval by microwaving, and then washed with PBS. Sections were incubated for 2 h at room temperature with anti-KI67 (ab279653, Abcam, USA), after which sections were rinsed with PBS, developed with DAB, counterstained with hematoxylin, cleared with xylene, and observed under a light microscope. A mean percentage of positive tumor cells was determined in at least five areas at × 400 and assigned to one of five of categories: (a) 0. < 5%; (b) 1. 5–25%; (c) 2. 25–50%; (d) 3. 50–75%; and (e) 4. > 75%. According to cell staining intensity score: (a) cell no color, 0 points; (b) straw colored, 1 point; (c) brownish-yellow, 2 points; and (d) tan, 3 points. Divided into 4 levels based on cell staining intensity score: (a) negative, 0-1points; (b) weak positive, 2-3 points; (c) positive, 4-5 points; (d) string positive, 6-7 points.

The tumors from the nude mice were fixed in 10% paraformaldehyde at 4°C for 12 h, and then dehydrated in different concentrations of ethanol, permeabilized using xylene and embedded in paraffin. The paraffin-embedded tumor tissues were then sliced (0.5 µm), rehydrated, and were stained with HE at 4°C for 10 min and sealed with neutral gum. For IHC assessment of E2F3 and Ki-67 in colorectal tumor tissues from the nude mice, the DAKO Envision system (Dako; Agilent Technologies, Inc.) was used. Briefly, after paraffin-embedded sections of tumor tissues were heated at 60°C, the sections were incubated with primary antibodies against E2F3 (1:200; cat. no. ab50917; Abcam) and Ki-67 (1:1,000; cat. no. ab279653; Abcam) overnight at 4°C. Then, the sections were incubated with biotin-labeled secondary antibodies (1:1,000; cat. no. ab205718; Abcam) at 37°C for 20 min. Under a light microscope, IHC staining scores for E2F3 were obtained by multiplying the intensity (0, negative; 1, low; 2, medium; and 3, high) with the extent of staining (0, 0%; 1, 0-10%; 2, 10-50%; 3, 50-75%; 4, >75%); the final scores were between 0-12. For evaluation of Ki67, the number of positive cells was calculated in three representative areas of high staining.

The excised tumor samples were embedded and fixed. Sections were microwaved in Retrieval Solution (Amyjet, Wuhan, China) at pH 6.0 or pH 9.0 for 7 min, followed by incubation in 3 % H₂O₂ for 10 min. Nonspecifically bound sites were then blocked with 2.5 % normal horse serum (Vector Biolabs, Burlingame, CA, USA). The sections were incubated with an antibody against Ki67 (#ab279653, Abcam, USA). After incubation with a secondary antibody (#ab125913, Abcam) for 30 min, the sections were stained with 3,3′-diaminobenzidine solution (Sangon Biotech, Shanghai, China) and hematoxylin (Sangon Biotech). Representative areas were viewed under a light microscope (Nikon 90i, Tokyo, Japan).