Differentiated fibrocytes were plated onto fibronectin (Sigma Aldrich) precoated slides after sorting by flow cytometry. Cells were fixed by 100% cold menthol and permeabilized with 1% Triton X‐100. The slides were blocked with 10% normal goat serum for 30 minutes. After blocking, 50 μL of diluted α‐smooth muscle actin antibody (1/200, Abcam) was applied overnight at 4°C. The secondary antibody, goat anti‐rabbit conjugated with Alex Fluro 488 (Invitrogen), was applied for 30 minutes in the dark. After washing, the slides were mounted with Prolong Gold Antifade reagent (Invitrogen).
α smooth muscle actin antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The α-smooth muscle actin antibody is a protein that specifically binds to and detects the presence of alpha-smooth muscle actin. This protein is commonly used as a marker for the identification and characterization of smooth muscle cells in various tissues and cell types.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Fibronectin, by Merck Group (1 mentions)
Alex fluro 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Sc 376764, by Santa Cruz Biotechnology (1 mentions)
St5020, by Leica (1 mentions)
F4 80 antibody, by Bio-Rad (1 mentions)
5 protocols using α smooth muscle actin antibody
1
Immunofluorescence Analysis of Differentiated Fibrocytes
2
Tissue Analysis for Cardiovascular Histology
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Heart and lung tissues were immersed in buffered 10% formalin at room temperature and were embedded in paraffin. Paraffin‐embedded tissues were cut and mounted on glass slides. Tissue sections were subjected to hematoxylin and eosin (H&E) stain, Verhoeff‐van Gieson stain, Masson's trichrome stain, and immunohistochemistry using the α‐smooth muscle actin antibody (Abcam, Cambridge, UK) or the platelet and endothelial cell adhesion molecule 1 antibody (sc‐376764, Santa Cruz Biotechnology, Dallas, TX).
3
Histological Analysis of Tissue Samples
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Tissues were immersed in buffered 10% formalin at room temperature and embedded in paraffin. Paraffin-embedded tissues were cut and mounted on glass slides. Tissue sections were subjected to hematoxylin and eosin (H&E) stain, Verhoeff-van Gieson stain, Masson’s trichrome stain, and immunohistochemistry with the α-smooth muscle actin antibody (Abcam, Cambridge, MA, USA).
4
Immunostaining of Sarcomeric and Smooth Muscle Actin
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Cells incubated on Lab-Tek chamber slides (Nalgene Nunc). The cells were fixed with 3% paraformaldehyde for 10 min at room temperature and washed with PBS. Cells were permeabilized in 0.5% Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH, pH 7.9, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) in PBS for 10 min and washed with PBS. They were blocked with PBS containing 0.3% goat serum and 5% bovine serum albumin for 1 hour at room temperature and then incubated for 1 hour with α-sarcomeric actin antibody (dilution 1∶200; Thermo) and α-smooth muscle actin antibody (dilution 1∶200; Abcam). Cells were washed with PBS and incubated with Alexa fluor 488 and rhodamine Red-X (dilution 1∶500; Invitrogen) as secondary antibody for 1 hour in dark room. After washing, the cells were mounted with ProLongantifade reagent containing DAPI. The immunoreactive signals were visualized by confocal laser scanning microscope LSM700 (Carl Zeiss, Germany).
5
Quantitative Macrophage Analysis in Tissues
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All collected tissues were fixed in 10% Neutral Buffered Formalin (NBF) for 24 h, processed to paraffin blocks, and cut into 4 micron sections. The sections were dried overnight in a 37 °C oven, followed by 1 hour incubation in a 60 °C oven prior to deparaffinization. Deparaffinization and H&E staining (Surgipath, Buffalo Grove, IL, USA) were performed on an automated multistainer (Leica ST 5020, Buffalo Grove, IL, USA). The F4/80 antibody, an IgG2b affinity purified rat monoclonal antibody, was purchased from AbD Serotec. The α smooth muscle actin antibody was purchased from Abcam. Morphometric analysis was performed using a Scan Scope XT (Aperio) and both Image Scope (Aperio) and Indica Lab (Indica Lab) software. For each animal, nine 3.6 × 105 μm2 areas were evaluated for the percent F4/80 positive staining per area of tissue.
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