Collagen biocoated slide chambers

Immunofluorescence was performed on ha-MSCs after osteogenic differentiation to detect MMP-9 protein. Briefly, 3 × 10⁴ cells were seeded on collagen-biocoated slide chambers (BD Bioscence); after 24 hours, cells were washed gently with PBS and fixed with cold absolute methanol, for 10 min at room temperature. Fixed cells were then blocked in 1% bovine serum albumin (BSA) in PBS solution and donkey serum, specific for the secondary antibody species, for 30 min at room temperature. After blocking, cells were incubated with primary MMP-9 antibody (1:500; Cell Signaling) for 1 hour at 37 °C. Samples were then washed with PBS, and incubated with Alexa Fluor 546 (1:250; Invitrogen) secondary antibody in 1% BSA/PBS for 1 hour at 37 °C in the dark. Finally, after washing, the samples were mounted and nuclei were counterstained with Pro Long anti-fade reagent with DAPI (Life Technologies). Images were acquired by a Leica DMI4000 B inverted fluorescence microscope (Leica Microsystems, Milan, Italy).

Immunofluorescence was performed on AAA-MSCs exposed to CoCl₂ to detect MMP-9 protein and its alteration following hypoxic induction. Briefly, 4 × 10⁴ AAA-MSCs were cultured on collagen biocoated slide chambers (BD Bioscence, San Jose, CA, USA) and after 24 h CoCl₂ was added to cultures according to the concentration range used for all the other experiments: 0–100 μM–500 μM for 24, 48, and 72 h. at the end of the treatment, cells were gently were washed with PBS and fixed with cold absolute methanol, for 10 min air dry at room temperature. Fixed cells were then blocked in 1% bovine serum albumin (BSA) in PBS solution and donkey serum, specific for the secondary antibody species, for 30 min at room temperature.
After blocking, cells were incubated with primary antibody anti-MMP-9 (1:500, Cell Signaling) for 1 h at 37°C. Samples were then washed with PBS and incubated with Alexa Fluor 546 (1:250; Invitrogen, Carlsbad, CA, USA) secondary antibody in 1% bovine serum albumin in PBS for 1 h at 37°C in the dark. Finally, after washes, the samples were mounted and nuclei counterstained with Pro Long anti-fade reagent with DAPI (Molecular Probes, Milan, Italy). Images were acquired by a Leica DMI4000 B inverted fluorescence microscope (Leica Microsystems, Milan, Italy) at × 20 magnification.