Ki 67 antibody

For apoptosis assay, BM cells were stained with antibodies for the surface markers, and incubated with the Annexin V-FITC antibody at room temperature for 15 minutes. DAPI (1 μg/ml) was added before analysis by FACS. For the in vitro apoptosis assay, the cells treated with the drug in vitro were directly stained with the Annexin V-FITC antibody, and DAPI was added before analysis by FACS. For caspase 3/7, 9 activity assays, HSPCs were plated into 96-well white luminescence plates and the Caspase3/7, 9 activities were analyzed according to the manufacturer’s instructions (Promega). For the mitochondrial membrane potential assay, the cells treated with the drug in vitro were determined by FACS after loading the cells with JC-1 dye according to the manufacturer’s instructions (Beyotime). For the BrdU incorporation assay, BrdU (100 mg/kg) was intraperitoneally injected, and followed by administration of BrdU (1 mg/ml) in the drinking water for 10 days. BrdU incorporation was determined by FACS analysis using the BrdU Flow Kit (BD Biosciences). For Ki67 staining, after the surface markers staining, the cells were fixed using BrdU Flow Kit and staining with Ki67 antibody (BD Pharmingen).

Cells were fixed in 4% paraformaldehyde (PFA), blocked with 5% goat serum in PBS-T (0.1% Tween 20 in PBS) for 1 h, and incubated overnight at 4°C with primary antibodies. Antibodies used were STAT3 and phospho-STAT3 (Abcam) and VEGFR2 (Cell Signaling). To assess cell proliferation, we used staining with Ki67 antibody (BD Bioscience). After washing, cells were incubated with respective Alexa 488 secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature, mounted in Vectashield antifade medium with DAPI (Vector Labs), and imaged with a confocal microscope (Nikon).

The sections of proximal jejunum were obtained from young and old mice sacrificed at 4 and 24 h post 14 Gy irradiation. The 5-μm-thick slides were deparaffinized, re-hydrated, and retrieved with antigen unmasking solution H-3300 (Vector, CA) in a steamer for 30 min. After 30 min of blocking with 10% normal serum from the same species, primary antibodies or corresponding control isotype IgG was applied to the slides and incubated overnight at 4 °C. Then, after incubation with biotinylated secondary antibodies and a VECTASTAIN ABC kit (Vector labs, PK-4000), the slides were developed with DAB detection and counterstained with hematoxylin (Vector, CA). For fluorescence staining, the slides were incubated with fluorochrome-conjugated secondary antibodies in the dark and counterstained with DAPI.
Ki67 antibody (BD Pharmingen, cat# 550609) was used at 2.5 μg/ml, coupled with biotinylated goat anti-mouse IgG (Vector, cat#BA-9200, 1:250). Primary cleaved caspase-3 (Asp175) (Cell Signaling, Ca#9661) was incubated at 1:100 dilution, followed by goat anti-rabbit IgG (Vector, cat #BA-1000, 1:1000 dilution).

A standard three-step streptavidin–biotin complex immunohistochemical assay was performed on paraffin sections for the detection of Ki-67 and phosphor-Histone H3 (Ser 10). Sections were deparaffinized and rehydrated in gradient concentrations of xylene and ethanol and then in distilled water. Sections were treated with 3% H₂O₂ to quench endogenous peroxidase activity. Antigen retrieval was performed by microwave pretreatment in 0.01 M citrate buffer for 25 min. After cooling, non-specific binding of the antibody to sections was blocked with 10% rabbit serum, Ki-67 antibody (BD#550609, Pharmingen, San Francisco, CA, USA) was applied in a 1:3000 dilution and incubated at 24 °C for 2 h. For pH3S10, antibody was diluted to 1:500. A subsequent incubation of 30 mins with biotinylated anti-rabbit serum was followed by a 30-min incubation using the Vectastain Elite ABC anti-rabbit kit (Vector Laboratories). The peroxidase was then developed in DAB (Diaminobenzidine, Amresco Inc. OH, USA) with H₂O₂. The sections were counterstained with hemotoxylin, dehydrated in gradient ethanol solution, cleared in xylene and mounted. One known liver tumor sample was included for positive control and for negative control when the primary antibody was replaced with 10% rabbit serum (manufacturer’s instructions for Vectastain Elite ABC anti-rabbit kit).