Dimethylformamide

To observe the expression levels, purity and immunoreactivity, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, MA), blocked by 6.25% non-fat dry milk for 1 h at room temperature. The membranes were probed with a 1/3333 dilution of monoclonal anti-polyhistidine antibody (Sigma-Aldrich, USA) for 1.5 h at room temperature. Then the membranes were probed with a 1/3333 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) antibody (Sigma-Aldrich, USA) for 1 h at room temperature. The blot was developed with diethanolamine buffer (10% Diethanolamine, 4 M HCl pH: 9.8, 0.5 mM MgCl2•6H2O) containing 4.3% 5-bromo-4-chloro-3-indolyl phosphate (BCIP) diluted in dimethylacetamide, 4.1% Nitro-BT diluted in 70% (v/v) dimethylformamide (Applichem, Germany).

In a modified version of the technique originally described by Kuznetsov et al. [37] (link), 10 million bone marrow cells were suspended in either 5 ml of normal medium (DMEM low glucose, 10% FCS, and 1% penicillin/streptomycin; all purchased from Gibco, Invitrogen) or osteogenic medium (normal medium, supplemented with dexamethasone [1 µl/ml] and ascorbic acid [50 µg/ml]; purchased from AppliChem, Germany). The medium was first changed after 72 h and then after every 3–4 days. On day 14, cells were washed with PBS and fixed in ethanol at 4°C for 20 min. Fixed cells were stained with alkaline phosphatase (ALP) staining solution (Tris, dimethylformamide, naphthol ASBI, and fast red; all purchased from AppliChem) and photographed. Cells were then discoloured by incubation with 90% ethanol overnight, stained with methylene blue (AppliChem), and then were photographed again.