Cells were cultured on 4-well chamber slide (BD Falcon, Bedford, MA, USA) and treated with or without TGF-β1 (R&D Systems, Abingdon, UK). After 24hrs of TGF-β1 (1 ng/mL) treatment, cells were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 15 min and followed by incubation with Protein Block for 15 min at room temperature. The cells were incubated with anti-α-SMA monoclonal antibody (Abcam, Cambridge, MA, USA) overnight at 4℃. The cells were washed with PBS and incubated with Alexa Fluor® 488 (Invitrogen, Carlsbad, CA, USA) secondary antibody for 1 hr at room temperature in dark room. After washing, the slides were mounted with Vecta-shield (Vector Labs, Burlingame, CA, USA) mounting media containing 4',6-diamino-2-phenylindole (DAPI) for detection of all nuclei. Fluorescence images were observed with a laser scanning confocal microscope (TCS SPE, Leica Microsystems GmbH, Wetzlar, Germany).
Anti α sma monoclonal antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-α-SMA monoclonal antibody is a laboratory reagent that can be used for the detection and localization of alpha-smooth muscle actin (α-SMA) in various biological samples. It is a highly specific and sensitive tool for identifying cells and tissues expressing this cytoskeletal protein.
Automatically generated - may contain errors
Lab products found in correlation
4 well chamber slide, by BD (1 mentions)
Tcs spe, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Penicillin streptomycin liquid, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by ZSGB-BIO (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 protocols using anti α sma monoclonal antibody
1
Quantifying TGF-β1-induced α-SMA Expression
2
Isolation and Hypoxia Preconditioning of hPVSMCs
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Human pulmonary vein smooth muscle cells (hPVSMCs) were aseptically isolated from the intrapulmonary vein (fourth level) from surgical pulmonary lobectomy at room temperature. After removing adhering fat, connective tissue, and endothelial cells, the dissected media of the PVs was cut into small pieces (1–2 mm2) and covered by autoclaved glass coverslips in cell culture dishes. Next, we cultured fourth level hPVSMCs in DMEM/F-12 (HyClone, USA) which was supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel), and 1% Penicillin-Streptomycin liquid (Gibco, USA) at 37°C and 5% CO2. PVSMCs were identified by positive immunostaining with anti-α-SMA monoclonal antibody (Abcam, UK). Cells at passages 2–3 were used in experiments, and each experiment was repeated at least three times with different preparations of cells. While the cells cultured in a tri-gas incubator, we performed hypoxia preconditioning (Forma Series II 3131 Water Jacket CO2 Incubator, Thermo Scientific, USA) for 12 or 24 h with oxygen concentration in 1% (Weisel, et al., 2014 (link)).
3
Quantifying Activated Hepatic Stellate Cells
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Immunofluorescence staining for α-SMA expression was performed to identify activated hPSCs. Specifically, 1×104 cells were seeded into each well of a 12-well plate. After treatment with MitoQ or vehicle for 48 h, the cells were fixed with 4% paraformaldehyde, and thereafter, blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) solution for 40 min. Next, the cells were incubated with anti α-SMA monoclonal antibody (#7817; Abcam, Cambridge, MA, USA) overnight at 4 °C followed by incubation with the secondary antibody (Alexa Fluor 594-conjugated goat anti-mouse IgG; ZSGB-BIO, Beijing, China) for 2 h. Nuclei were then stained with DAPI (ZSGB-BIO, Beijing, China), and the quantity of α-SMA positive cells was determined by analyzing the images obtained via fluorescence microscopy (Leica, Wetzlar, Germany) using Image J software (National Institute of Health (NIH), Bethesda, MD, USA).
4
Immunohistochemical Analysis of α-SMA
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Paraffin-fixed liver sections were incubated with Animal Research Kit Peroxydase (Dako, Carpinteria, CA). As the primary antibody, we used anti-α-SMA monoclonal antibody (dilution 1:200; Abcam, Cambridge, UK). Next, the slices were incubated with horseradish peroxidase-conjugated streptavidin and diaminobenzidine, and then counterstained with hemalaun. As negative controls, primary antibody was replaced with nonimmune immunoglobulin at the same concentration. No staining was observed in the negative controls.
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