Cardiac fibroblasts were cultured on glass coverslips at 2 × 104 cell/glass in a 24-well plate. After treatment with TGFβ and EVs, cells were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 25 min. Cells were then washed with PBS and blocked with 10% FBS at room temperature for 30 min. Next, the cells were incubated with anti-actin, α-smooth muscle-Cy3 (Sigma-Aldrich) or anti-col1A (Cell Signaling Technology) at a concentration of 1/200 in a humidified incubator overnight. After washing in PBS with PBS, and in the case of col1A treated with the Alexa488 Donkey anti-rabbit (BD Bioscience) for 1 h, the cells were washed with PBS and counterstained with DAPI for 15 min and mounted using a drop of FluorSave (CalbioChem, San Diego, CA, United States). Images were captured with a Leica DM2500 fluorescence microscope.
α smooth muscle cy3
Manufactured by Merck Group
α-smooth muscle-Cy3 is a fluorescently labeled antibody that binds to α-smooth muscle actin, a protein found in the contractile apparatus of smooth muscle cells. It can be used to visualize and identify smooth muscle cells in various tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin, by Merck Group (2 mentions)
Anti col1a, by Cell Signaling Technology (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
Pe mouse igg1 p3, by Thermo Fisher Scientific (1 mentions)
Pe cd39 5f2, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by BD (1 mentions)
Atp bioluminescence assay kit cls 2, by Merck Group (1 mentions)
A1r microscope, by Nikon (1 mentions)
Anti laminin, by Merck Group (1 mentions)
Anti activated caspase 3, by Cell Signaling Technology (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using α smooth muscle cy3
1
Immunocytochemistry of Cardiac Fibroblasts
2
Histological Analysis of Atherosclerosis in Aorta
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This experiment was performed according to methods reported previously.19 , 20 En face pinned aortas were stained with Oil Red O. Images of the aortas were captured with a Nikon digital camera. The proximal aortas attached to the heart were embedded in OCT compound and frozen at −80°C. The aortic root was serially sectioned into 8‐μm sections from the site where the aortic valve appears. A set of 6 consecutive sections were stained with Oil Red O and Masson's Trichrome for morphological analysis of the atherosclerotic plaque. For immunofluorescence, macrophages or the expression of VCAM‐1 in the aortic root were detected by staining sequential sections with rat anti–Monocyte macrophage (MOMA‐2) monoclonal antibody or rabbit polyclonal anti‐VCAM, followed by the corresponding secondary antibodies conjugated with Alexa Fluor 488 or 568 for fluorescence detection. Smooth muscle actin cells were detected with α‐Smooth Muscle–Cy3 (1:400; Sigma‐Aldrich). Images were captured using an Axio Imager M1 (Zeiss). Quantitative analysis of the sections was performed by a technician who was blinded to the treatment group of the mice using Image‐Pro Plus 6.0 software (Media Cybernetics).
3
Isolation and Characterization of Murine Vascular Smooth Muscle Cells
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VSMCs were isolated from murine aorta as previously described (27 (link)). In order to isolate enough cells, three to four aortas were pooled together. Briefly, aortas were dissected and the adventitial tissue and fat were removed. Then aortas were cut into 1–2 mm rings and put in digestion with Collagenase type II (Gibco) for 6 h (7.5 mg of Collagenase II in 5.5 ml of culture medium: DMEM containing 10% FBS, 1× penicillin/streptomycin, and 1% glutamine). The lysate was pelleted and the cells were resuspended in 700 μl of culture medium and cultured at 37°C, 5% CO2. After 5 days, the medium was replaced and, once confluent, cells were trypsinized and expanded. Cells were cultured up to the third passage. The purity of VSMCs was checked by immunofluorescence staining with anti-actin, α-Smooth Muscle-Cy3 (1A4, #C6198, 1:5,000; Sigma-Aldrich). Cd39 expression was evaluated by FACS analyzing 30,000 cells using PE Cd39 (5F2, #12-3390-80, 1:100; eBioscience) and PE mouse IgG1 (P3.6.2.8.1, #12-4714-41, 1:100; eBioscience) as isotype control.
4
Quantifying Tumor Angiogenesis, Apoptosis, and Proliferation
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Breast and colon tumors were fixed in 4% paraformaldehyde (Merck) and included in OCT blocks. Frozen 15-μm sections were incubated with fluorescein-labeled GSL I-B4 (5 µg ml−1, Vector Laboratories), anti-CD31 (1:200, BD Pharmingen), anti-Actin, α-Smooth Muscle-Cy3 (1:100, Sigma) and anti-Laminin (1:25, Sigma) to assess angiogenesis, anti-activated caspase-3 (1:200, Cell Signaling) to assess cell death and anti-Ki67 (1:250, Thermo Fisher Scientific) to assess proliferation. Nuclei were counter stained with DAPI (diamidino-2-fenilindol) reagent. Cellular fluorescence was analyzed by confocal microscopy in a Nikon A1R+ microscope. The images were processed with ImageJ v1.46 software.
Aliquots of freeze-clamped tumor powder were used to measure total ATP content using the ATP Bioluminescence Assay Kit CLS II (Sigma-Aldrich) following the manufacturer’s instructions.
Aliquots of freeze-clamped tumor powder were used to measure total ATP content using the ATP Bioluminescence Assay Kit CLS II (Sigma-Aldrich) following the manufacturer’s instructions.
5
Immunofluorescence Analysis of Atherosclerotic Plaques
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