Rabbit anti ki67 antibody

Immunohistochemistry staining of tumor tissues was performed using the routine procedures. The rabbit-anti Ki-67 antibody (1: 400 dilution; Thermo Fisher Scientific) and HRP-conjugated secondary goat-anti-rabbit monoclonal antibody (1: 200 dilution; Thermo Fisher Scientific) were used. Before being mounted, the slices were stained with 0.5% diaminobenzidine and counterstained with Mayer's hematoxylin.

Coronal sections (40 μm thick) were cut through the entire DG of the left or right (randomized) hippocampus with a vibratome (Leica VT 1000 S). All brain sections were collected sequentially in a 6-well plate, which was filled with cryoprotectant solution (30% sucrose + 30% ethylene glycol in 0.1 M phosphate buffer, pH 7.4). The samples were stored at -20°C until staining.
The sections were first incubated with 2 N HCl at room temperature for 1 hour. Thereafter, the sections were neutralized with 0.1 M borate buffer and then incubated with a rat anti-BrdU antibody (1:100, Accurate Chemical and Scientific Corporation, Westbury, NY, USA) at 4°C overnight. After extensive washing, the sections were incubated with a donkey anti-rat secondary antibody conjugated to Alexa 488 (Molecular Probes) at room temperature for 2 hours. Ki67 immunostaining was conducted with a rabbit anti-Ki67 antibody (1:200, Thermo Fisher Scientific, Rockford, IL, USA) and a donkey anti-rabbit secondary antibody conjugated to Alexa 555 (1:200, Thermo Fisher Scientific, Rockford, IL, USA).
Images were captured using an Olympus FV1000 Viewer confocal microscope (Tokyo, Japan). Every 6^th section throughout the DG was analyzed for the number of BrdU+ cells and Ki67+ cells, which were counted by an investigator who was blinded to the treatment.

Cryosections were washed in PBS and then blocked for 1 h at room temperature in blocking solution (PBS containing 5% normal serum and 0.1% Tween‐20). After removal of the blocking solution, the sections were reacted overnight at 4°C with rat anti‐mouse CD31 antibody (1:400 dilution; BD Biosciences), rat anti‐mouse F4/80 antibody (1:100 dilution; AbD Serotec, Raleigh, NC, USA), mouse anti‐α‐smooth muscle antibody (1:100 dilution; Sigma‐Aldrich), or rabbit anti‐Ki67 antibody (1:100 dilution; Thermo Fisher Scientific Inc.) 4°C followed by Alexa 488‐ or Alexa 594‐conjugated secondary antibodies (1:2000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and DAPI for 1 h at room temperature. Finally, the specimens were mounted with fluorescent mounting medium (DAKO Corporation, Carpinteria, CA, USA) and images were acquired by using a fluorescence microscope (DMI6000B; Leica Microsystems, Wetzlar, Germany).

Cryosections (10 μm thick) of paraformaldehyde-fixed ileum and colon tissue of GF and CR mice were stained for proliferative cells using rabbit-anti-Ki-67 antibody (Thermo Scientific, #RM-9106-S1, Waltham, MA, USA) and Rabbit IgG Vectastain Elite ABC kit (Vector Labs, #PK-6101, Burlingame, CA, USA). Sections were counterstained with hematoxylin/eosin. Ki-67-positive cells were counted for 10 crypts per section from eight mice per group.