For histological examination, murine lungs were inflated by 1.2 mL 4% paraformaldehyde (PFA) (Electron Microscopy Sciences), and then transferred to a 50-ml polypropylene tube containing 10 mL 4% PFA. After 24-h of fixation at 4 °C, the lobes of the mouse lungs were separated, and transferred to tissue cassettes (Tissue-Tek). The tissue cassettes were then immersed in 70% ethanol at 4 °C. Primary antibodies against vimentin (1:100, Cell Signaling Technology, 5741), α-smooth muscle actin (1:640, Cell Signaling Technology, 19245), and collagen III (1:1000, Abcam, ab7778) were used for IHC staining. For IHC staining, the paraffin-embedded lung sections were first baked and deparaffinized. To retrieve antigen, the slides were heated on the Bond III Autostainer at 99–100 °C, and the sections subjected to sequential incubation with an endogenous peroxidase block, primary antibody, secondary antibody, polymer, diaminobezidine, and hematoxylin. Finally, the sections were dehydrated in 100% ethanol, and mounted in Cytoseal XYL (Richard Allan Scientific). Appropriate positive and negative controls were included.
α smooth muscle actin
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
α-smooth muscle actin is a protein that is a key component of the contractile apparatus in smooth muscle cells. It plays a crucial role in the regulation of smooth muscle contraction and is widely used as a marker for smooth muscle cells in various research applications.
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Lab products found in correlation
β actin, by Cell Signaling Technology (6 mentions)
Luciferase, by Novus Biologicals (2 mentions)
Tsa fluorescein plus kit, by Leica Biosystems (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
F4 80, by Abcam (1 mentions)
Gs 800 imaging densitometer, by Bio-Rad (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Resistin, by Abcam (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Blocking buffer, by Bio-Rad (1 mentions)
Ab283575, by Abcam (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Bolt bis tris plus 4 12 sodium dodecyl sulfate polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Digital camera, by Zeiss (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
14 protocols using α smooth muscle actin
1
Histological Examination of Murine Lungs
2
Immunohistochemical Analysis of Skin Samples
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Skin biopsies were fixed in 3.7% formalin overnight for paraffin embedding. For frozen sections, samples were fixed in 3.7% paraformaldehyde at 4°C for 1 h, rinsed in PBS, sunk in 30% sucrose overnight and embedded into OCT. Frozen sections were probed with antibodies against the following antigens: p21 (1:100, Cell Signaling) and p16 (1:100, Invitrogen). Paraffin sections were antigen-retrieved by boiling slides in 1 mM EDTA, pH 8.0, for 10 min, and probed with antibodies against the following antigens: K14 (1:1,000, Biolegend), Ki67 (1:100, Cell Signaling and BD Biosciences), α smooth muscle actin (1:500, Cell Signaling), cleaved caspase-3 (1:100, Cell Signaling) and luciferase (1:1000, Novus). Staining was amplified using the TSA Fluorescein Plus kit for antibodies against the following targets: p53 (1:5000, Novocastra), NICD (1:500, Cell Signaling) and Mycn (1:500, Cell Signaling). Amplification was performed for 2 min, 4 min and 10 min, respectively, following manufacturer’s instructions. Image processing was performed using Adobe Photoshop with the Auto-Blend feature applied to maximize image sharpness across focal planes.
3
Western Blot Analysis of Liver Proteins
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Protein extract preparation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein transfer to polyvinylidene difluoride membrane and antibody labelling were completed as previously described [23 (link)]. The membranes were immunoblotted with primary antibodies to sterol regulatory element-binding protein-1c (SREBP-1c) (C-20), Acetyl CoA Oxidase (ACO) (Santa Cruz Biotechnology, Santa Cruz, CA), fatty acid synthase, FoxO1, phospho-Akt (p-Akt; Ser473), phospho-AMP kinase-α (p-AMPK-α; Thr172), phospho-NF-κB p65 (Ser536), α-smooth muscle actin (Cell Signaling Technology, Danvers, MA), or F4/80 (Abcam, San Francisco, CA), with β-tubulin (Cell Signaling Technology, Danvers, MA) or Ponceau staining used to control for protein loading. The HRP-conjugated secondary antibody was used to detect the protein bands, which were imaged by scanning densitometry with a model GS-800 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA) after visualization with chemiluminescence reagent (AmershamTM ECLTM Prime Western Blotting Detection Reagent). The bands of interest were quantified using AlphaView SA software (Protein Simple, Santa Clara, CA), and their intensities were expressed relative to the intensity of suitable loading control.
4
Protein Expression Analysis in Heart and Fibroblasts
5
Protein Expression Assay in Gastric Cancer
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The tissues or human gastric cancer cells were washed with phosphate-buffered saline (PBS). Total protein samples were extracted, and protein concentrations were measured using the Bio-Rad Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Equal quantities of total proteins were separated through BOLT BISTRIS PLUS 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Thermo Scientific, New York, NY, USA) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with a blocking buffer (Bio-Rad, Hercules, CA, USA) for 30 min at room temperature and incubated with the primary antibodies SOX2 (1:1000; #23064; Cell Signaling, Danvers, MA, USA), GRP78 (1:1000; #3183; Cell Signaling, Danvers, MA, USA), β-actin (1:5000; #4967; Cell Signaling, Danvers, MA, USA), CREB3L1/OASIS (1:1000; ab137565, Abcam, Cambridge, UK), matrix metallopeptidase 9 (MMP-9; 1:1000; ab283575, Abcam, Cambridge, UK), and α-smooth muscle actin (1:1000; #14968; Cell Signaling, Danvers, MA, USA) at 4 °C after the membranes were washed with PBS with Tween 20. The membranes were incubated with secondary antibodies at room temperature for 1 h and then analyzed using an electrochemiluminescence detection system.
6
Immunofluorescence Analysis of EMT Markers
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H292, H292-shCTL, H292-shDUOX1 and H292-E90 cells were seeded in eight-well glass chamber slides, fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin (BSA) in PBS for 15 min. Slides were washed 2 × with 1% BSA/PBS solution, blocked with 10% goat serum for 1 h and then subjected to staining with antibodies against E-cadherin (1:100; Invitrogen), vimentin (1:100; Cell Signaling) or α-smooth muscle actin (1:100; Cell Signaling). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (10 μg/ml in 1% BSA/PBS). Cells were washed 2 × with 1% BSA/PBS and mounted with glass coverslip. Images were taken on a Zeiss LSM 510 META laser scanning confocal microscope (Zeiss, Jena, Germany). Brightfield images were taken using a Zeiss Interskop phase contrast microscope interfaced with a digital camera (Zeiss).
7
Experimental Lung Fibrosis Model
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The reagents included bleomycin (Nippon Kayaku Ltd., Tokyo, Japan), saline (Kelun Pharmaceutical Co., Sicuan, China), Pirfenidone (Beijing Continent Pharmaceuticals Co., Ltd, Beijing, China), Hydroxyproline assay kit instructions (#A030-2-1, Nanjing Jian Chen Bioengineering Institute, China), protease inhibitor (Roche Life Science, USA), BCA protein assay kit (BIO-RAD, USA), polyvinylidene fluoride (PVDF) membranes (Millipore, Germany), skim milk (Beyotime, China), chemiluminescence detection kit (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Scientific, USA), enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, Austria), lysing solution (Merck, Germany). Primary antibodies used in our project were as follows: α-Smooth Muscle Actin (Cell Signaling Technology, #19245, USA), anti-collagen I (Cell Signaling Technology, #81375, USA), β-Actin or glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, #5174, USA), anti-CD3-PerCP (Invitrogen, Austria) anti-CD4-FITC (Biolegend, USA), anti-CD8-APC (Biolegend, USA). All horseradish peroxidase–conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
8
Protein Extraction and Western Blot
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Protein extractions were performed and 15 μg of extracted proteins were separated by SDS–PAGE and revealed by antibodies directed against actin (1:1000, Cell Signaling Technology, #8457), PDGFRα (1:1000; Cell Signaling Technology, #3174), and α-smooth muscle actin (1:1000, Cell Signaling Technology, #19245).
9
Western Blot Analysis of SMAD3 and α-SMA
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Standard methods were used to perform western blotting using antibodies against phosphorylated SMAD3, total SMAD3, α-smooth muscle actin (α-SMA), and β-actin from Cell Signaling (Danvers, MA, USA).
10
Immunohistochemical Analysis of Skin Samples
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