Cells ± RTV infection were seeded onto chamber slides in BM + additives for HCE-TJ or EMEM + 10% serum for HCF. When ~60–80% confluent, media was removed, and the cells were rinsed 3 times with PBS, starved overnight (BM or EMEM only), incubated in BM or EMEM ± 2ng/ml of TGF-β1 for 20 minutes, fixed with methanol, and then processed for IF [24 (link)]. In brief, slides were blocked with blocking solution (1% bovine serum albumin [BSA] + 0.1% Triton-X in phosphate buffered serum [PBS]), incubated overnight at 4°C with anti-Smad2 (Abcam), anti-α-smooth muscle actin (SMA: DAKO; Carpinteria, CA) or anti-Ki67 (Vector Labs; Burlingame, CA), and then incubated for 1 hour at room temperature with the appropriate secondary antibody conjugated to fluorescein (Jackson ImmunoResearch; West Grove, PA). Slides were coverslipped with mounting media containing DAPI (Vector Labs), which is a nuclear counter stain. Slides were observed with a Nikon Eclipse E800 for IF and DIC (differential interference contrast) and photographed with either an Andor Clara E camera with Nikon NIS Elements for Basic Research or SPOT digital camera (Micro Video Instruments). Samples where the primary antibody was omitted served as negative controls.
Anti α smooth muscle actin sma
Manufactured by Agilent Technologies
Anti-α-smooth muscle actin (SMA) is a protein that is commonly used as a marker for smooth muscle cells. It is used in various laboratory applications to identify and quantify these cells.
Automatically generated - may contain errors
2 protocols using anti α smooth muscle actin sma
1
TGF-β1 Induced Smad2 Activation Assay
2
Immunocytochemical Analysis of Muscle Progenitors
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Cells and tissue were fixed in 2% PFA and processed for histology and immunocytochemistry as previously described (Gargioli et al., 2008 (link)). Briefly, MP were cultured in fibronectin-coated 8-well chamber slides (Nunc) as described above. After fixation with 2% PFA for 10 min, cells were incubated overnight at 4°C with the following primary antibodies: anti-α-smooth muscle actin (SMA; Dako), platelet-derived growth factor receptor beta (PDGFR-β; Cell Signaling Technology), chondroitin sulfate proteoglycan (NG2; Merk Millipore) and myosin heavy chain (MyHC; DHSB), diluted in accordance with the manufacturers' instructions. Cells were then incubated with the appropriate secondary fluorophore-conjugated antibody. Nuclei were stained with DAPI. Fluorescence photomicrographs were taken with an Axio Observer A1 microscope equipped with a fluorescence detection system at 20 × magnification. Imaging analysis was performed with AxioVision Imaging System software (Zeiss).
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