Mouse monoclonal α sma antibody

The cells were washed with cold PBS, harvested with a cell lifter, centrifuged and lysed with RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na deoxycholate and 0.1% SDS) supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis IN), homogenized with a pellet pestle motor (Kimble/Kontes, Vineland, NJ) and subsequently centrifuged at 13,000 x g for 15 min at 4°C. For Western blots either aliquots of culture media containing the proteins secreted by the cultured cells or aliquots of the cell lysates containing cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Blots were blocked for 1 h in Tris-buffered saline(TBS)-Tween (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat dry milk (BioRad, Hercules, CA). The membranes were then incubated overnight at 4°C with mouse monoclonal α-SMA antibody (Abcam, 1:200), and GAPDH polyclonal rabbit antibody (Abcam, 1:2000) in a 5% nonfat dry milk/TBS-Tween solution. Membranes were then washed with TBS-Tween, and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, UK) diluted 3000-fold in 5% nonfat dry milk/TBS-Tween. The signals were quantified using NIH Image J software.

For IF on cultured isolated HC- and SSc-FBS cells were grown on 8-well culture slides (BD) maintained in DMEM medium (Sigma) supplemented with 10%FBS (Sigma) and 100 units/ml penicillin, and 100 ng/ml streptomycin (Sigma) at 37 °C in a humidified atmosphere of 5%CO₂. FBS were fixed in 4% buffered paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS. Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam). The immunoreaction was revealed using the appropriate biotinylated secondary antibodies and the signal was amplified with a streptavidin AlexaFluor 488 coniugate (Invitrogen). Cell nuclei were visualized using DAPI. Fluorescence was analyzed using an Olympus BX53 fluorescence microscope (Fig. 2C).