Tumor sections were stained with hematoxylin–eosin to visualize tumor morphology. Immunohistochemical evaluation was performed using primary monoclonal anti-smooth muscle actin antibody (1:100, Dako) and EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse (Dako, Glostrup, Denmark). All images were taken using a Leica DMC5400 fluorescence microscope with a 10× objective with the Leica LAS X image acquisition software.
Anti smooth muscle actin antibody
Manufactured by Agilent Technologies
Sourced in Belgium
The Anti-smooth muscle actin antibody is a laboratory reagent used to detect and visualize smooth muscle actin in biological samples. It is a targeted antibody that specifically binds to smooth muscle actin, a key structural protein found in the cytoskeleton of smooth muscle cells.
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4 protocols using anti smooth muscle actin antibody
1
Tumor Morphology and Smooth Muscle Analysis
2
Histological Assessment of Absorb BVS
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Between August 2013 and January 2015, the pathology department of the University of Amsterdam Academic Medical Center received a total of 4 lesions treated with 5 Absorb BVS, with duration of implantation ranging from 3 to 501 days. All available clinical records were reviewed for patient history, duration of implantation, risk factors, medications, and cause of death. The necessity to obtain informed consent was waived by the institutional review committee.
All autopsies and histological assessments were performed by dedicated cardiovascular pathologists. The treated arteries were dissected from the heart and submitted for plastic embedding in methyl methacrylate. Histological sections were cut at 6 μm and stained with Hematoxylin (Klinipath, Duiven, the Netherlands) and Eosin (Merck, Darmstadt, Germany) for overall histomorphology, elastic van Gieson stain (Klinipath, Duiven, the Netherlands) for elastin and collagen fibers, and Alcian blue stain (Sigma, St. Louis, USA) for proteoglycans. Additional immunohistochemical stains with anti–smooth muscle actin antibody and an anti‐CD31 antibody (DAKO, Heverlee, Belgium) were applied in all cases for visualization of smooth muscle cells and endothelial cells, respectively.
All autopsies and histological assessments were performed by dedicated cardiovascular pathologists. The treated arteries were dissected from the heart and submitted for plastic embedding in methyl methacrylate. Histological sections were cut at 6 μm and stained with Hematoxylin (Klinipath, Duiven, the Netherlands) and Eosin (Merck, Darmstadt, Germany) for overall histomorphology, elastic van Gieson stain (Klinipath, Duiven, the Netherlands) for elastin and collagen fibers, and Alcian blue stain (Sigma, St. Louis, USA) for proteoglycans. Additional immunohistochemical stains with anti–smooth muscle actin antibody and an anti‐CD31 antibody (DAKO, Heverlee, Belgium) were applied in all cases for visualization of smooth muscle cells and endothelial cells, respectively.
3
Isolation of Smooth Muscle Cells from Saphenous Vein
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SMC were isolated from freshly isolated SV segments using standard explant techniques [18 (link)]. SV segments were denuded of their endothelium, stripped of their adventitia, cut into ~ 1 mm squares, and placed on tissue-culture treated plastic and feed with culture medium. Once cells had migrated out of the tissue, the tissue was removed and discarded. Smooth muscle cell phenotype was confirmed by immunocytochemical staining with an anti-smooth muscle actin antibody (Dako). SMC from passages 3–8 were used.
4
Immunohistochemical Analysis of Tissue Samples
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Sections were deparaffinized and incubated overnight with 0.1 M Tris/HCL buffer (pH 9.0) at 80°C. Next day, samples were incubated at room temperature (RT) for 30 min and washed with PBS three times. This was followed by H 2 O 2 treatment (15 min) to remove endogenous peroxidase activity. Afterwards, sections were washed with PBS three times and incubated with 10% serum from the species that produced secondary antibodies. Subsequently, sections were incubated with primary antibody; anti-smooth muscle actin antibody (Dako Heverlee, Belgium) or ED-1 (anti-CD 68) antibody (Bio-Rad Puchheim, Germany). Bound antibodies were detected with peroxidase-conjugated rabbit-anti-mouse IgG (Dako, Glostrup, Denmark). Staining was performed with 3, 3'-Diaminobenzidine (DABStained samples were scanned by Nano-Zoomer digital slide scanner (Hamamatsu, Japan) and quantified with Aperio ImageScope software (Leica, the Netherlands).
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