Mouse monoclonal vimentin

Immunofluorescence studies were performed for the detection of intracellular Vimentin protein in the MSC samples. Trypsinised cells were seeded on coverslips kept in a 35 mm dish (BD Biosciences, USA), and MSC expansion media were added and incubated at 37°C, 5% CO_2. Once confluency reached within 40–50%, the existing media from the dish were removed and cells were washed with PBS. Cells were fixed with 1 ml of 4% paraformaldehyde for 20 mins at 4°C. Permeabilization and blocking was done with 0.05% Tween-20 followed by 2% BSA in PBS for 30 mins at room temperature. MSCs were incubated with the Mouse monoclonal vimentin (Abcam, Cambridge, USA) primary antibody (1/100) for overnight at 4°C. Next day, after washing with PBS, cells were incubated with the secondary fluorescent antibody (1/500) for 40 mins at room temperature (RT). To visualise nuclei, slides were stained with diluted 1/4000 DAPI (Life Technologies, USA) for 3 mins at RT followed by thorough washing of the cells with PBS. The acquisition and imaging of the cells were performed using Floid Microscopy (Life Technologies).

Protein extracted from cells was separated on 10% SDS-polyacrylamide electrophoresis gels and transferred to a polyvinylidene difluoride membrane and immunoblotted with specific antibodies. The primary antibodies included mouse monoclonal STAT3, rabbit monoclonal phosphorylated (p-STAT3), α-smooth muscle actin (α-SMA; Sigma, St. Louis, MO, USA), monoclonal rat IL-6, rabbit GAPDH, and mouse monoclonal vimentin (Abcam, Cambridge, MA, USA). Anti-GAPDH antibody was used as a sample-loading control. Immunoreactive protein bands were detected with the ECL detection system.