Cleaved caspase 3 primary antibody

For the immunohistochemical studies, the sections were incubated overnight at 4 °C with the monoclonal rabbit anti-mouse primary antibody (PCNA, 1/500; Abcam, Cambridge, CA, USA) and the polyclonal cleaved caspase-3 primary antibody (1/200, Abcam, Cambridge, CA, USA). Then, the sections were rinsed with 0.01 mol/L PBS (pH 7.4) and incubated with biotinylated sheep anti–rabbit IgG (1/300; Sigma, Cambridge, CA, USA) for 2 h at room temperature. After washing, the tissues were incubated with streptavidin–horseradish peroxidase (1/300, Sigma, St. Louis, MO, USA) for 2 h and 30 min at room temperature. Immunoreactivity was visualized by incubating the tissue sections in 0.01 mol/L PBS containing 0.05% 3′,3–diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO, USA) and 0.003% hydrogen peroxide for 10 min in the dark. The sections were then stained with hematoxylin and mounted. The images were acquired on an upright DP72 microscope (Olympus, Tokyo, Japan). Image analysis system (Image-pro plus 6.0) was used to count the number of PCNA–positive cells and measure the mean integral optical density (IOD) of cleaved caspase-3 positive cells. The data were counted from 10 random fields from three cross-sections of the three specimens from each group.

HP-MSCs were seeded on a 96 well plate with glass bottom, fixed in 100 % methanol for 10 min, permealized with 0.5 % Triton X-100 in PBS for 10 min, incubated with cleaved caspase-3 primary antibody for twenty-four hours (1:500, Abcam, Cambridge, UK), then washed, and the secondary antibody anti-rabbit IgG (1:1000) was added for 1 h. The presence of cleaved caspase-3 immune-positive HP-MSCs was identified and quantified in five images for each group using Image-Pro Plus program.