Ki 67 monoclonal antibody

Tumor tissues were fixed, dehydrated, embedded in paraffin, and serially sectioned at a thickness of 4 μm. Then the sections were incubated with primary and secondary antibodies. Briefly, the paraffin‐embedded tissue was dried, dewaxed, and rehydrated. Then the sections were rinsed phosphate with buffered saline solution (PBS, Beyotime, Shanghai, China) and blocked with bovine serum albumin (Beyotime, Shanghai, China). The sections were incubated with Ki-67 monoclonal antibody (34,330, Cell Signaling Technology, MA, USA) and cle-caspase3 (9664, Cell Signaling Technology, MA, USA) at 4 °C overnight. The sections were incubated with a secondary antibody the next day for 30 min at 37 °C. The diaminobenzene was used as the chromogen, and hematoxylin was used as the nuclear counterstain. The IHC scores were obtained by multiplying the percentage of positive tumor cells and the intensity of staining. The value of positive tumor cells was defined as 0 (no staining), 1 (0%–25%), 2 (25%–50%) and 3 (> 50%). The value of intensity of staining was defined as 0, 1, 2 and 3^{25 (link)}.

Ileal samples were sectioned at 5 μm, placed in 10 mM citrate buffer of pH 6.0, and heated for 10 min. Sections were incubated for 10 min in 3% hydrogen peroxide (Sigma-Aldrich), washed with distilled water and phosphate-buffered saline for 5 min each, blocked in 10% normal horse serum in PBS for 1 h at room temperature (RT), and then incubated with one of the primary antibodies overnight at 4°C: cleaved caspase-3 (Asp175) monoclonal antibody or Ki67 monoclonal antibody (Cell Signaling, Boston, MA). After being washed, sections were incubated with conjugated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 1 h at RT, washed, and incubated for1 h at RT with R.T.U. Vectastain Elite ABC reagent, according to the manufacturer's protocol (Vector Laboratories). After the washings, the section was developed with a 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories), to give a brown color. Slides were dehydrated in serial ethanol and xylene solution and permanently mounted. Images were digitally captured at ×200 using an Olympus BX51 microscope, and quantification of cleaved caspase-3 and Ki67 staining was performed in a blind manner by counting positive cells in multiple random microscope fields per tissue section.