The antibodies against VEGFR3 (goat anti-mouse, R&D Systems (Minneapolis, MN) #AF743) and VEGFR2 (goat anti-mouse, R&D Systems #AF644) were used at a 1:50 dilution; the anti-LYVE (rabbit anti-mouse Abcam,Cambridge, MA) #ab14917) was used at 1:250; the anti-αSMA (mouse anti-mouse Dako,Santa Clara, CA) #M0851) was used at 1:100, and anti-PECAM/CD31 antibody (rat monoclonal, catalog #DIA-310; Dianova, Hamburg, Germany) was used at 1:250. Secondary antibodies were Donkey anti-rabbit 594 (#21207 ThermoFisher, Grand Island, NY); Donkey anti-goat 488 (Abcam #ab150129); Donkey anti-goat 546 (ThermoFisher #A11056); and Donkey anti-mouse 488 (Abcam #ab150105) were all used at a dilution of 1:250. Sections from a total of 3 pairs of A10ΔEC mice and littermate controls were analyzed per antibody, with at least one pair of littermates of each gender.
Anti α sma
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom
Anti-α-SMA is a monoclonal antibody used for the detection of alpha-smooth muscle actin (α-SMA) in immunohistochemistry and Western blot applications. α-SMA is a cytoskeletal protein expressed in smooth muscle cells and is commonly used as a marker for various cell types, including myofibroblasts and vascular smooth muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Anti vimentin, by Agilent Technologies (3 mentions)
Anti α tubulin, by Merck Group (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Anti vwf, by Agilent Technologies (2 mentions)
Envision dual link system hrp kit, by Agilent Technologies (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti col1a1, by Rockland Immunochemicals (2 mentions)
Bz 9000, by Keyence (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Ab150105, by Abcam (1 mentions)
Af743, by R&D Systems (1 mentions)
Af644, by R&D Systems (1 mentions)
Donkey anti goat 488, by Abcam (1 mentions)
A11056, by Abcam (1 mentions)
Gal 3, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Anti col1a1, by Abcam (1 mentions)
Anti pdgfrβ, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
32 protocols using anti α sma
1
Immunofluorescence Staining of Vascular Markers
2
Evaluation of Kidney Injury in Leptospirosis
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Nephritis was graded blindly by a pathologist on a scale of 0–4 in a whole longitudinal section of the organ following previously published criteria for scoring kidney injury present in leptospirosis [31] (link). The PS technique was carried out as previously described [60] (link), [61] (link), [62] (link). Digital image analysis was used to quantify the amount of red-stained collagen fibers as previously described [28] (link) using a Nikon E200 microscope with a Tucsen TCC 5.0 digital camera and the software provided by the manufacturer. The IHC procedure have been previously described [43] (link) using anti α-SMA, (Clone 1A4, Dako), Gal-3 (Clone M3/38), MAC (Abcam 55811) and an anti LipL32 (a gift from Dr Nascimento, Butantan Institute). Acute necrotizing pancreatitis was used as a positive control for the MAC IHC [63] (link), [64] (link).
3
Blood Vessel Assessment in Grafted Ovaries
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The endothelial and pericyte cells were stained to identify new blood vessels in
vitrified/grafted ovaries (n=3 from each group). The 5μm paraffin sections (three serial
sections from each sample) were deparaffinized with xyloland rehydrated in graded alcohol
series (Merck, Germany). The sections were incubated with hydrogen peroxide (3%)
inmethanol for 30 minutes at room temperature to block endogenous peroxidase. After
autoclaving in the citrate buffer, the slides were incubated with the primary antibodies
for 30 minutes:1/100 antivWF (Dako, Denmark) for staining endothelial cells, and 1/100
anti-α-SMA (Dako, Denmark) for staining smooth muscle cells. The slides were washed with
PBS, and stained with the EnVision+Dual Link System HRP kit (Dako, Denmark),
3-Diaminobenzidine (DAB).
Each specimen was observed under a light microscope
(×400) (Nikon, Japan). Single or clusters of endothelial
cells positive for yWF (brown dye), were considered
indicative of vessels formation. In the current experiment,
the results from treatment groups were compared with
those of the intact ovarian tissue from the control mice.
All immunohistochemical analyses were done in three
independent experiments.
vitrified/grafted ovaries (n=3 from each group). The 5μm paraffin sections (three serial
sections from each sample) were deparaffinized with xyloland rehydrated in graded alcohol
series (Merck, Germany). The sections were incubated with hydrogen peroxide (3%)
inmethanol for 30 minutes at room temperature to block endogenous peroxidase. After
autoclaving in the citrate buffer, the slides were incubated with the primary antibodies
for 30 minutes:1/100 antivWF (Dako, Denmark) for staining endothelial cells, and 1/100
anti-α-SMA (Dako, Denmark) for staining smooth muscle cells. The slides were washed with
PBS, and stained with the EnVision+Dual Link System HRP kit (Dako, Denmark),
3-Diaminobenzidine (DAB).
Each specimen was observed under a light microscope
(×400) (Nikon, Japan). Single or clusters of endothelial
cells positive for yWF (brown dye), were considered
indicative of vessels formation. In the current experiment,
the results from treatment groups were compared with
those of the intact ovarian tissue from the control mice.
All immunohistochemical analyses were done in three
independent experiments.
4
Western Blot Analysis of Fibroblast Markers
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Cells were washed with ice-cold PBS and scraped with ice-cold lysis buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, 0.2 mM dithiothreitol, and 0.5 % NP-40) supplemented with protease (Roche Diagnostics, Mannheim, Germany) and phosphatase (Roche Diagnostics) inhibitors. Protein concentrations were determined using the BCA protein assay kit (Pierce Chemical Co, Rockford, IL, USA). Fifteen microgram of protein was separated on a 8 % Tris-glycine SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes (Amersham Biosciences, Little Chalfront, UK) using a wet blotting apparatus (Mini Trans-Blot Cell, BioRad, Nazareth, Belgium). Blots were blocked with 5 % milk powder in Tris buffered saline (TBS) with 0.2 % Tween (Sigma) and subsequently incubated overnight with primary anti-PDGFRβ (diluted 1/1000 in blocking buffer) (Abcam), anti-αSMA (1/50) (Dako), anti-COL1A1 (1/1000) (Abcam), or anti-GAPDH (diluted 1/30,000) (Abcam). The membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1/20,000) (Dako, Glostrup, Denmark) for 1 h, and the antigen was visualized by enhanced chemiluminescence using ECL substrate (Pierce Chemical Co.).
5
Immunophenotyping of Pluripotent Stem Cells
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The cells were fixed with 4% paraformaldehyde, and treated with PBS containing 5% normal goat or donkey serum, 1% BSA, and 0.2% TritonX-100. The following antibodies were used: SSEA3 (1:10), TRA-1-81 (1:50), TRA-1-60 (1:50) (these antibodies were used at Kyoto University and were kind gifts from Dr. Peter W. Andrews), anti-SSEA4, anti-TRA-1-81, anti-TRA-1-60 (1:500, all contained in the ES Cell Characterization Kit from Merck Millipore; these antibodies were used at Gifu University), anti-NANOG (1:20, R&D Systems), anti-OCT3/4 (1:1000, Santa Cruz Biotechnology), anti-βIII-tubulin (1:200, Cell Signal Technology), anti-βIII-tubulin (1:2000, Covance), anti-α-SMA (1:500, DAKO), and anti-AFP (1:100, R&D). Nuclei were stained using 1 μg/mL Hoechst33342 (Life Technologies).
6
Immunohistochemical Detection of α-SMA
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On deparaffinized sections, the antigenic sites were unmasked with Tris buffer EDTA (ethylenediamine tetra-acetic acid) at pH 9 for 30 minutes. Immunostaining was performed using the PLC BenchmarkTX (Ventana) and XT ultra13 kit view diaminobenzidine (Ventana). The negative control was performed without the primary antibody.
Slides were incubated with PBS containing mouse monoclonal antibody anti-α-SMA diluted at 1/300 (Dako) at 4 °C overnight. The sections were then incubated with anti-mouse antibody conjugated with peroxidase-labeled polymer (Dako). Immunoreactive proteins were visualized with a 3-amino-9-ethylcarbazole-containing peroxidase substrate (hydrogen peroxide) (Dako). Finally, the tissue sections were counterstained with hematoxylin.
Slides were incubated with PBS containing mouse monoclonal antibody anti-α-SMA diluted at 1/300 (Dako) at 4 °C overnight. The sections were then incubated with anti-mouse antibody conjugated with peroxidase-labeled polymer (Dako). Immunoreactive proteins were visualized with a 3-amino-9-ethylcarbazole-containing peroxidase substrate (hydrogen peroxide) (Dako). Finally, the tissue sections were counterstained with hematoxylin.
7
Immunohistochemical Analysis of Lung Tissue
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Immunohistochemistry was performed at McMaster’s Histology Core Facility as previously described15 (link). Briefly, formalin-fixed tissues were cut at 5 µm sections and stained with Masson’s trichrome for ECM, anti-αSMA (Dako, cat# M0851) for the identification of αSMA-positive myofibroblasts and anti-fibronectin (Abcam, cat# ab2413) for the assessment of pro-fibrotic factors. Lung tissue sections were also stained with anti-arginase-1 (BD Biosciences, cat# 610708) for the identification of alternatively activated macrophages. We used the Dako ARKTM (Animal Research Kit) Peroxidase (cat# K3954) to execute immunohistochemical stains involving mouse primary antibodies on mouse lung tissues. As for tissues that contain endogenous biotin (such as liver and kidney and other tissues), a step involving avidin/biotin block (Vector Laboratories, cat# SP2001) was conducted prior to immunohistochemical staining of the lung tissues. Diluent and IgG negative controls as well as non-treated tissue controls were included to ensure precision of the staining protocol and optimal antibody concentrations.
8
Comprehensive Immune Cell Profiling Protocol
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The following primary antibodies and factors were used in this study: anti‐vitronectin (ab45139, Abcam, Cambridge, MA; MAB38751, R&D Systems, Inc., Minneapolis, MN), anti‐thrombospondin 1 (Ab‐11, Thermo Scientific, Hudson, NH; ab1823, ab3131, Abcam), anti‐FX (H‐120 and C‐20, Santa Cruz Biotechnology, Santa Cruz, CA), anti‐fibrinogen (CSI19761A, Cell Sciences Inc., Canton, MA), anti‐albumin (A90‐134A, Bethyl Laboratories, Inc., Montgomery, TX), anti‐CD45 (ab10558, Abcam; M0701, DAKO, Carpinteria, CA), anti‐CD11b (BD Biosciences, San Jose, CA), anti‐MECA32 Ab (BD Biosciences), anti‐αSMA (M0851, DAKO), anti‐CD4 (RM4‐5, BD Biosciences), anti‐CD8a (53–6.7, BioLegend, San Diego, CA), anti‐CD11c (HMα2, BioLegend), anti‐B220 (RA3‐6B2, BioLegend), anti‐NK‐1.1 (PK136, BioLegend), anti‐TCRβ (H57‐597, BD Biosciences) and anti‐IFNγ antibodies (Santa Cruz Biotechnology). CCL2, VEGF, TNFα, G‐CSF and SDF1 were purchased from R&D Systems (Minneapolis, MN). IL‐6 and CXCL1 (Miltenyi Biotec, Bergisch Gladbach, Germany), TGF‐β (Peprotech) and HGF Wako Pure Chemical Industries) were used.
9
Signaling Pathways Activated by Estrogen and Cytokines
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All reagents were from Sigma‐Aldrich (Poole, UK) unless otherwise stated. Primary antibodies and dilutions for Western blot and immunocytochemistry were polyclonal rabbit anti‐p‐EGFR (1:1000), anti‐p‐ERK1/2, anti‐p‐STAT1, anti‐p‐ STAT3, anti‐STAT1, and anti‐STAT3 (all at 1:2000) from Cell Signalling Technology Inc. (Beverly, MA, USA), polyclonal mouse anti‐GAPDH (1:5000) from Abcam (Cambridge, UK), and anti‐αSMA (1:50) from DAKO (Aachen, Germany). All RT–QPCR reagents were obtained from Life Technologies (Paisley, UK). Other reagents used were E2 (Sigma‐Aldrich) and recombinant human IFNγ and TGF‐β1 (R&D Systems, Abingdon, UK).
10
Quantitative Analysis of Cytoskeletal Proteins
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After extraction, content determination and electrophoresis on a 5–20% gradient gel, 50 μg of proteins were transferred to nitrocellulose filters (Schleicher and Schuell, Dassel, Germany) and incubated with anti-α-SMA (1:250; DAKO) and anti-fibrillin-1 (1:200; Merck Millipore, Massachusetts, USA) followed by a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; Sigma Aldrich, Saint Louis, MI, USA). Normalization was performed using anti-α-tubulin (Sigma Aldrich, Saint Louis, MI, USA). Detection and quantification were carried out in triplicate experiments, as reported [11 (link)].
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