We fixed cells in 24-well plates with 4% Paraformaldehyde (PFA, Sigma-Aldrich) at 37°C for 10 minutes and rinsed cells with phosphate buffered saline (PBS) two times for 30 minutes. PBS with 0.01 M Glycine and 0.1% Triton-X was placed on the cells for 30 minutes. Cells were then rinsed with 5% BSA/PBS and 1% BSA/PBS followed by incubation with primary antibodies against SMα-actin (αSMA) (Sigma-Aldrich), calponin (Abcam) and SM myosin heavy chain (SM-MHC) (Abcam) at 4°C. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (Millipore) was added the following day to detect the localization of anti-calponin antibodies while FITC-conjugated goat anti-mouse secondary antibody (Millipore) was used to detect the localization of antiαSMA and anti-SM-MHC antibodies. Images were visualized using a Nikon Eclipse TE2000-S fluorescence microscope (Nikon USA, Melville, New York, USA)
Smα actin αsma
Manufactured by Merck Group
Sourced in United States
SMα-actin (αSMA) is a protein found in the contractile apparatus of vascular smooth muscle cells. It is commonly used as a marker for the identification and quantification of smooth muscle cells in various research applications.
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2 protocols using smα actin αsma
1
Immunofluorescence Analysis of Smooth Muscle Markers
2
Isolation of Rat Pulmonary Arterial Smooth Muscle Cells
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The primary PASMCs were isolated from the pulmonary arteries of male Sprague-Dawley rats (5 weeks old) by using a tissue-sticking method. After the dissection of adventitia and endothelia, pulmonary arteries were quickly cut into small pieces and stuck to cell culture bottle. The primary PASMCs were confirmed by immunofluorescent stainings for SM α-actin (α-SMA) and Desmin (Sigma-Aldrich, St. Louis, MO, USA). The basic culture medium was consisted of DMEM-F12 supplemented with 5% fetal bovine serum (FBS) while the starvation medium was with 1% FBS. The primary PASMCs between passage 3 and 6 were used for experiments.
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