Ab 59545

For microscopy analyses, the hBMSCs were cultured on glass coverslips and treated according to experimental groups described earlier. After 3 days, cells were washed with PBS, fixed in PBS-paraformaldehyde 4% for 1 h and permeabilized in PBS containing 0.2% Triton-X 100 and 1% BSA at 37°C for 1 h and stained with primary antibodies diluted according to the manufacturer’s instructions: mouse monoclonal OCT4 (ab-59545, Abcam; 1:100 dilution) and mouse monoclonal NANOG (1E6C4—sc-293121, Santa Cruz Biotechnology; 1:100 dilution) for 1 h at room temperature. After washing with PBS, the cells were stained with secondary antibody rabbit anti-mouse IgG H+L Alexa Fluor 488 (Invitrogen) for 1 h at room temperature. Cells were washed with PBS and coverslips were mounted on glass slides using Fluoroshield with DAPI (Sigma) and then viewed on Zeiss Axio Observer inverted microscope with Apotome.2 (Zeiss, Germany). The quantification of NANOG and OCT4 was performed as previously described [25 (link)].

The protein extracts were obtained and extracted in 500 μL Laemmli Buffer [SDS 4%, glycerol 20%, Tris-Cl (pH 6.8) 120 mM, bromophenol blue 0.02% (w/v) and DTT 0.1 M] and samples were stored at -20°C until further analyses. 15 mL protein (75 mg) was resolved by SDS-PAGE and blotted onto Immobilon FL PVDF membranes (Millipore, Bedford, MA, USA), blocked in Tris-buffered saline (TBS) with 0.05% Tween 20, albumin 2,5% (TBSTA) and incubated overnight at 4°C with NANOG (1E6C4—sc-293121, Santa Cruz Biotechnology) and OCT4 (ab-59545, Abcam) antibodies, followed by the appropriate horseradish peroxidase (HRP)-linked secondary antibody, at 1:5000 dilution, in TBSTA for 1 h. The immunoreactive bands were detected with enhanced chemiluminescence kit.