Liver samples were fixed in 10% neutral formalin fixative and embedded in paraffin, and were sectioned at 4 µm thickness. Histological analysis was performed in hematoxylin-eosin staining and Sirius red staining. For immunohistochemistry, liver sections were incubated with anti-ARK5 (Santa, Santa Cruz, CA, USA, sc-271828), anti-α-SMA (Invitrogen, Waltham, MA, USA, PA5-18292), anti-collagen I (Invitrogen, Waltham, MA, USA, PA5-29569) primary antibodies overnight at 4 °C. Positive staining was visualized with the EnVision immunodetection system (K5007; Dako, Copenhagen, Denmark) according to the manufacturer’s instructions. For immunofluorescence, liver sections or cells were fixed in ice-cold methanol and were incubated with anti-ARK5 (Santa, Santa Cruz, CA, USA, sc-271828), anti-α-SMA (Invitrogen, Waltham, MA, USA, PA5-18292), anti-p-Smad2/3 (Abcam, Cambridge, UK, ab254407), anti-Smurf2 (Santa, Santa Cruz, CA, USA, sc-518164), anti-TβRI (Santa, Santa Cruz, CA, USA, sc-518018), anti-Smad4 (Cell Signaling Technology, Beverly, MA, USA, 46535S) and anti-HNF-4α (Abcepta, Suzhou, China, AP60014) antibodies followed by incubation with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA). Nucleis were counterstained with DAPI. Images were captured using an inverted fluorescence microscope or confocal laser scanning microscope.
Anti α sma
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-α-SMA is a primary antibody that detects alpha-smooth muscle actin (α-SMA), a cytoskeletal protein found in smooth muscle cells. It is commonly used in immunohistochemistry and western blotting applications to identify and quantify the presence of α-SMA in various tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Fv3000, by Olympus (2 mentions)
Anti collagen 1, by Thermo Fisher Scientific (1 mentions)
Anti ark5, by Thermo Fisher Scientific (1 mentions)
Anti p smad2 3, by Abcam (1 mentions)
Anti smad4, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 or 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd31 alexa488, by BioLegend (1 mentions)
Anti cd26, by R&D Systems (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Anti atgl, by Cell Signaling Technology (1 mentions)
Anti hsl, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ecl plus western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Carl Roth (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
Goat anti mouse alexa 647, by Thermo Fisher Scientific (1 mentions)
28 protocols using anti α sma
1
Histological and Immunofluorescence Analysis of Liver
2
Immunofluorescence Analysis of Lung Tissue
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Lungs were embedded in Tissue-Tek®,O.C.T. Compound (SAKURA Finetek, Tokyo) and frozen in liquid nitrogen for preparation of cryosections. Frozen lung tissues were cut into 6 μm thick sections, immunostained and visualized by confocal microscopy (Fluoview FV 10i, Olympus, Tokyo). The sections were fixed in acetone for 10 min, blocked with Block Ace (Dainippon Sumitomo Pharma, Tokyo) for 10 min, and incubated with the primary and secondary antibodies for 60–120 min. The following antibodies were used for immunostaining: anti-CD31-Alexa488 (BioLegend, San Diego, CA), anti-CD45-Alexa647 (BioLegend), anti-CD26 (R&D Systems, Minneapolis, MN), anti-α-SMA (Thermo Scientific, Waltham, MA), and anti-S100 calcium-binding protein A4 (S100A4) (Abcam, Cambridge, UK).
3
Immunoblotting Analysis of Protein Expression
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Analysis of protein expression by immunoblotting was performed as previously described (10 (link)). Proteins of cell lysates (20–40 μg) were dissolved in SDS sample buffer, separated by 10% or 12.5% SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (Carl Roth GmbH, Karlsruhe, Germany). The membrane was blocked with 10% nonfat dry milk and incubated with the following primary antibodies: anti-ATGL, anti-HSL, anti-GAPDH, and anti-calnexin from Cell Signaling Technology (Danvers, MA; 2138S/ATGL, 4107S/HSL, 2118S/GAPDH, 2679S/calnexin), anti-6x HIS tag, and anti-NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) from Abcam (Cambridge, England; ab18184/6x HIS-tag®, ab157221/NDUFS1), anti-α-SMA from Thermo Fisher Scientific (Waltham, MA; PA5-22251/α-SMA), and anti-KIAA1363 from Invitrogen GmbH (PA5-50285/NCEH1 = KIAA1363), respectively. For detection, membranes were incubated with horseradish peroxidase-labeled secondary antibodies specific for respective primary antibody. Bands were visualized using the ECL plus Western blotting Detection Reagent (Thermo Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).
4
Visualizing Lipid Droplets and α-SMA
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To visualize LDs and α-SMA, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences Hatfield). Subsequently, cells were stained with DAPI and LD540 (kindly donated by Dr C. Thiele) (50 (link)). Staining for immunofluorescence was performed with anti-α SMA (Thermo Fisher Scientific), followed by goat-anti-mouse-alexa647 or donkey-a-rabbit-alexa647 (Life Technologies). Cells were mounted with FluorSave (Calbiochem) and subsequently imaged with a Leica TCS SPE Laser Scanning Spectral Confocal Microscope using preset settings for the representative dyes.
5
Immunohistochemical Analysis of Apoptosis
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Sections were stained with anti-BAX (Cell Signaling, Danvers, MA, USA), anti-cleaved caspase-3 (ABCAM), anti-α-SMA (ABCAM), or CD68 (Thermo Scientific) at room temperature for 2 hours. TUNEL solution and secondary antibodies (PE-conjugated goat anti-rabbit IgG and PE-conjugated goat anti-mouse IgG [Santa Cruz Biotechnologies]) were then added and sections incubated for 1 hour at 37°C in the dark. TUNEL assays were performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Sections were mounted using Fluoroshield with DAPI and confocal microscopy was performed with a LSM 700 system (Carl Zeiss).
6
Immunohistochemical Analysis of Corneal Tissue
7
Endothelial Cell Protein Expression Changes
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HUVECs were exposed to the EP4 antagonist (AH23848; 100 μM, Sigma, Kanagawa Prefecture, Japan) with or without L-902,688 (1 µM) or TGF-β (5 ng/mL) for 24 h. Western blotting was performed using anti-eNOS (BD), anti-E-cadherin (E-cad) (Cell Signaling, Danvers, MA, USA), anti-CD146 (Abcam, Cambridge, UK), anti-α-SMA (Thermo, Waltham, MA, USA), and anti-Twist (Novus, Frauenfeld, Switzerkand) primary antibodies. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling) were administered as secondary antibodies. Blots were visualized using the enhanced chemiluminescence detection system (Amersham, UK). Samples were normalized to GAPDH (Santa Cruz, CA, USA) and quantified by densitometry.
8
Comprehensive Molecular Signaling Pathway Analysis
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Anti-NOX-2, anti-Rac-1, anti-p47phox, anti-p-53, anti-Bax, anti-Bcl-2, anti-cytochrome c, anti-β-actin, anti-p-I-κBα, anti-p-p38, anti-p-NF-κB, anti-COX-2, anti-IL-8, anti-TGFβ1, anti-p-ERK, anti-Sp1, anti-CTGF, anti-FGF2, anti-uPA, anti-MMP-2, anti-MMP-9, and anti-α-SMA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA).
9
Immunohistochemical Analysis of Liver Tissue
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Liver tissue samples were recovered, fixed in 10% buffered formalin, and embedded in paraffin. Sections (4 μm) were dewaxed and hydrated through graded ethanol, cooked in 25 mM citrate buffer, pH 6.0, in a pressure cooker for 10 min, transferred into boiling deionized water, and let to cool for 20 min. Tissue sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity (Tieppo et al., 2009 (link)). The slides were incubated with rabbit anti-α-SMA, CLOCK, and REV-ERBα (Thermo Fisher Scientific, Rockford, IL, United States) polyclonal antibodies overnight at 4°C. Subsequently, the sections were incubated for 30 min using the EnVision1 system and developed with a solution of 3-3-diaminobenzidine (Vector Lab, Burlingame, CA, United States). The specificity of the technique was evaluated by negative controls (omitting the incubation with the primary antibody and incubating it with non-immune sera). Positive areas were quantified using the ImageJ software v3.91 (NIH, Bethesda, MD, United States).
10
Multiparametric Flow Cytometry Analysis of Mouse Lung Cells
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Mouse lung cells were pretreated with anti-CD16/32 antibody (BioLegend) to block Fc receptors, and then incubated with specific antibodies at 4 °C in the dark. The following antibodies were used for cell surface staining: anti-CD31-PE/Cy7 (BioLegend), anti-CD45-Alexa700 (BioLegend) and anti-CD26-PE (BioLegend). To measure α-SMA and S100A4 levels, after surface staining the cells were incubated with anti-α-SMA (Thermo Scientific) and anti-S100A4 (Abcam) for 35 min at 22 °C. Cells were then incubated for 25 min at 22 °C with donkey anti-rabbit IgG-Alexa 488 (IgG; H + L) (Life Technologies) as a secondary antibody.
HMVEC-Ls were pretreated with anti-CD16/32 antibody and then incubated with anti-CD31, −CD45, and -α-SMA. Cell fluorescence was measured using a FACSCantoTM II instrument (Becton Dickinson, San Jose, CA) and the output was analyzed with FlowJo software (Tree Star, San Carlos, CA).
HMVEC-Ls were pretreated with anti-CD16/32 antibody and then incubated with anti-CD31, −CD45, and -α-SMA. Cell fluorescence was measured using a FACSCantoTM II instrument (Becton Dickinson, San Jose, CA) and the output was analyzed with FlowJo software (Tree Star, San Carlos, CA).
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