Mouse monoclonal anti human vinculin igg

The cell area was analyzed for 30 cells for each condition after 24 h of culture using ImageJ (version 1.53e). For cytoskeleton analysis, hUC-MSCs were plated at an initial cell density of 1.5 × 10⁴ cells/well onto the glass coverslips, which were clean (control) or covered by different PEM films. The cells were cultured for 24 h and fixed in 3.7% formaldehyde, solubilized with 0.1% Triton X-100, immunostained with mouse monoclonal anti-human vinculin IgG (Merck, Darmstadt, Germany) and Alexa Fluor 488-conjugated goat anti-mouse IgG-clone A11001 (Merck, Darmstadt, Germany), and counterstained with TRITC-phalloidin (Merck, Germany). Nuclei were stained with DAPI (Merck, Germany). The specimens were mounted onto coverslips with poly(vinyl alcohol) (Dako Fluorescent Mounting Medium, Agilent Technologies, Santa Clara, CA, USA), visualized under a fluorescence inverted microscope (Axio Vert.A1, Zeiss, Dresden, Germany), and analyzed using ZEN 2.3 (Zeiss) software.

hUC-MSCs were seeded on coverslips coated with a (DR/HP)₆ multilayer without proteins (control) and with proteins, at a density of 1.5 × 10⁴ cells/well in a 12-well culture plate. The cells were cultured in DMEM/F12 medium with 10% FBS for 24 h. Next, they were fixed using the standard protocol in warm 3.7% formaldehyde for 15 min, solubilized with 0.1% Triton X-100 in PBS for 7 min, and washed in PBS. Then, cells were immunostained with mouse monoclonal anti-human vinculin IgG in PBS solution of 3% BSA (Merck, Darmstadt, Germany). Alexa Fluor-488-conjugated goat anti-mouse IgG-clone A11001 (Merck, Darmstadt, Germany) was counterstained with TRITC-phalloidin (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Nuclei were stained with DAPI following the manufacturer’s protocol (Merck, Darmstadt, Germany). The specimens were mounted onto coverslips with poly(vinyl alcohol) (Dako Fluorescent Mounting Medium, Agilent Technologies, Santa Clara, CA, USA). An inverted fluorescence microscope (Axio Vert.A1, Zeiss, Dresden, Germany) was used to visualize the cells which were analyzed using ZEN 2.3 (Zeiss, Dresden, Germany) software.