Rhodamine conjugated goat anti mouse igg

Cells were exposed to 0.5 μm of ADR or left untreated. Twenty-four hours after treatment, cells were washed twice in ice-cold PBS, fixed in 3.7% folmaldehyde in PBS at room temperature for 30 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and then blocked with 3% BSA in PBS at room temperature for 1 h. After blocking, cells were washed in ice-cold PBS and simultaneously incubated with mouse monoclonal anti-RUNX2 (8G5; Medical & Biological Laboratories, Nagoya, Japan) and rabbit polyclonal anti-p73 (Bethyl Laboratories, Montgomery, TX, USA) antibodies at room temperature for 1 h followed by the incubation with rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. After the extensive washing, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK). Fluorescent images were captured using a confocal microscope.

To assess the osteogenic differentiation of TSPCs on membranes at the protein level, immunofluorescent staining assays for Collagen I alpha 2 (Col1a2), Osteocalcin (Ocn) and Runx-2 (n=3 in each group) were performed at day 7, 14 and 21 after co-culture in vitro. Briefly, the samples were fixed in 4% paraformaldehyde for 15 min and washed with PBS three times for 5 min each. The samples were permeabilized by incubation with 0.1% Triton-X100 in PBS for 10 min followed by incubation in 3% BSA in PBS for 1 h to reduce nonspecific background staining. Next, these samples were treated with a primary antibody overnight at 4°C, incubated with a secondary antibody for 1 h at room temperature and then counterstained with DAPI (KeyGEN, China) for 20 min under dark conditions. Antibodies were used at the following concentrations: Anti-Col1a2 antibody (ab96723; Abcam, UK), 1:150; Anti-Ocn antibody (ab13420; Abcam, UK), 2 μg/mL; Anti-Runx-2 antibody (ab192256; Abcam, UK), 1:1000; FITC-conjugated goat anti-rabbit IgG, 1:500 (Invitrogen, USA); Rhodamine-conjugated goat anti-mouse IgG, 1:500 (Invitrogen, USA). The stained samples were visually observed using LSCM (Zeiss LSM710, Carl Zeiss, Germany).