The heart tissues were fixed in 10% neutral buffered formalin after excision and weighing and processed using standard techniques. Five μm histological sections were prepared and stained with hematoxylin-eosin (H&E) and Masson’s Trichrome stains. Immunohistochemical staining was performed using antibodies for F4/80 (1:200, Abd serotec Raleigh, NC), collagen III (1:20, SouthernBiotech, Birmingham, AL), alpha SMA (1:500, Abcam, Cambridge, MA), iNOS (1:800, Abcam, Cambridge, MA) and CD206 (1:800, Abcam, Cambridge, MA). All measurements and quantifications were performed in a random blinded fashion using Olympus Bx50 microscope (Olympus Optical Co. Ltd., Buffalo Grove, IL), Micropublisher 3.3 RTV camera (QImaging, Surrey, BC, Canada). The amount of necrosis in each heart section was assessed on H&E sections. Quantitative analysis of % area of fibrosis for trichrome and % positively stained area for F4/80, alpha SMA and collagen III was performed using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200X magnification. The number of iNOS+ cells was counted at 200X and CD206+ cells at 400X high field magnification.
Alpha sma
Manufactured by Abcam
Sourced in United Kingdom
Alpha-SMA is a protein found in smooth muscle cells. It 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
Nis elements br 4.13.00 64 bit image analysis system, by Nikon (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
Collagen 3, by Southern Biotech (1 mentions)
Cd206, by Abcam (1 mentions)
Bx51 fluorescent microscope, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
37 c incubator, by Thermo Fisher Scientific (1 mentions)
Movas, by American Type Culture Collection (1 mentions)
Benchmark xt, by Roche (1 mentions)
Cd163, by Novus Biologicals (1 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Desmin, by Agilent Technologies (1 mentions)
5 protocols using alpha sma
1
Histological and Immunohistochemical Analysis of Heart Tissue
2
Characterization of MOVAS Cell Line
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The mouse aorta smooth muscle cell line (MOVAS) was purchased from American Type Culture Collection (ATCC) center (Menassas, VA, USA). MOVAS were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, USA), containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Sigma-Aldrich, USA).Cells were incubated in humidified 5% CO2, 37 °C incubator (Thermo, MA, USA) and measured by a hemocytometer. Cells were co-stained by alpha-SMA (Abcam, Cambridge, UK) and DAPI (Bioworld Biotechnology, Minnesota, USA), and imaged with an Olympus BX51 fluorescent microscope (Olympus, Tokyo, Japan).
3
Immunohistochemistry of Liver Tissue Samples
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Immunohistochemical staining of formalin fixed paraffin-embedded liver sections was performed using a panel of antibodies including CD3, CD8, CD20, CD68 (Dako), alpha-SMA (Abcam), and CD163 (Novus Biologicals). Briefly, sections of 3 to 5 μm were deparaffinized through graded alcohols and xylene. Immunohistochemical staining was performed after antigen retrieval using either citrate buffer (10 mmol, pH 6.0) or EDTA (1 mmol, pH 9.0). Slides were incubated in Tris-goat serum (3%) for 15 min and then incubated at room temperature with primary antibodies. Detection was carried out on the automated system BenchMark XT autostainer (Ventana Medical Systems) according to the manufacturer’s instructions. Images were taken using an Olympus BX41 microscope, objective UPlanFI 20×/0.75, with an adaptor U-TV0.5xC using a digital camera Q-imaging Micropublisher 5.0 RTV. The images were captured using Q-Capture version 3.1.
4
Immunocytochemical Protein Analysis
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Immunocytochemistry was performed using polyclonal antibodies for PARP, ki67, alpha-SMA, and vimentin in a working dilution of 1:500 (Abcam Cambridge, MA). The slides were then stained with either donkey anti-rabbit or donkey anti-mouse horseradish peroxidase-linked secondary antibodies (1:500 dilution). The stained slides were examined in a Leica microscope under high power (x40).
5
Immunostaining and TUNEL Assay Protocol
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