Paraffin-embedded heart sections were evaluated for ECM content by staining with Masson’s trichrome and specific immunostains for collagen type-I alpha1 (Sigma), and αSMA (Biolegend), as well as phospho-Smad2 (EMD Millipore), phospho-p38, and phospho-JNK (Cell Signaling) staining for these signaling pathways. Nuclei were stained with DAPI (Life Technologies). Quantitation of phospho-stained nuclei was performed using ImageJ.
α sma
Manufactured by BioLegend
Sourced in United States
α-SMA is a protein marker commonly used in immunohistochemistry and western blotting applications to detect the presence of smooth muscle actin in biological samples. It is a key component of the contractile apparatus in vascular smooth muscle cells and myofibroblasts.
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Lab products found in correlation
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Masson s trichrome, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab133357, by Abcam (1 mentions)
Anti galectin 3, by BioLegend (1 mentions)
Picrosirius red psr, by Merck Group (1 mentions)
Col1a1, by Abcam (1 mentions)
Visiomorph, by Visiopharm (1 mentions)
Catalase, by Abcam (1 mentions)
Enhanced chemiluminescence substrate, by Bio-Rad (1 mentions)
Vimentin, by BioLegend (1 mentions)
Tnf α, by Abcam (1 mentions)
E cadherin, by BioLegend (1 mentions)
Fuji blue x ray film, by Bio-Rad (1 mentions)
Klotho, by Abcam (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
5 protocols using α sma
1
Cardiac Extracellular Matrix Analysis
2
Histological Assessment of Liver Tissue
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Remaining paraffin-embedded liver tissue was subsequently cut into 3 μm sections at Gubra (Hørsholm, Denmark). Sections were stained with hematoxylin-eosin (HE), picro-Sirius Red (PSR, Sigma-Aldrich, Brøndby, Denmark), anti-galectin-3 (cat. 125402, Biolegend, San Diego, California, USA), alpha-smooth muscle action (α-SMA, cat. ab124964, Abcam, Cambridge, UK), anti-type I collagen (COL1A1, cat. 1310–01, Southern Biotech, Birmingham, Alabama, USA), CD11b (cat. ab133357, Abcam, Cambridge, UK) or CD45 (cat. ab10558, Abcam, Cambridge, UK) using standard procedures. A digital imaging software (Visiomorph®, Visiopharm, Hørsholm, Denmark) was used for quantification of whole-section liver fat (HE-staining), fibrosis (PSR, COL1A1), inflammation (galectin-3, CD11b, CD45) and hepatic stellate cell activation (α-SMA). Histochemical positive staining areas were expressed relative to total tissue sectional area (fractional area, in percent).
3
Quantitative Protein Expression Analysis
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The protein expression levels were determined by Western blotting. The samples were re-labeled for a blind analysis before the Western blot processing. Equal amounts of protein extracts were separated using sodium dodecyl sulfate (SDS)-PAGE and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies for E-cadherin (#866702), vimentin (#677802), α-SMA (#904601) (Biolegend, San Diego, CA, USA), p53 (#sc-126), p16 (#sc-377412), p21 (#sc-6246), β-actin (#sc47778), GAPDH (#sc25778) (Santa Cruz Dallas, TX, USA), catalase (#ab16731), superoxide dismutase 1 (SOD1; #ab13498), Klotho (#ab203576), CD68 (#ab125212), TNF-α (#ab183218) (Abcam, Cambridge, MA, USA), Ly6G (#14-5931-82) (eBioscience, San Diego, CA, USA), Bax (#14796), Bcl-2 (#3498), cleaved Caspase 3 (#9664), and CHOP (#2895) (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The blot signals were measured using enhanced chemiluminescence substrates (Bio-Rad) and developed onto a Fuji Blue X-ray Film. ImageJ software was used to quantify the protein expression bands.
4
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections were deparaffinized in xylene, hydrated in a graded series of ethanol, and blocked by incubation with 1% BSA in PBS for 2 h. The following primary antibodies were used at 4 °C overnight at the indicated dilutions: α-SMA (1:100; Biolegend); IL-6 (1:30; Invitrogen); Ki67 (SP6; 1:200; Abcam); pan-cytokeratin (AE1/AE3 + 5D3; 1:200; Abcam); E-cadherin (1:200; R&D Systems, Wiesbaden, Germany); N-cadherin (1:200; BD Transduction Laboratories, Franklin Lakes, NJ, USA); and collagen type 1 (1:200; Invitrogen). After washing with PBS, sections were incubated for 2 h at room temperature in secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:400; Alexa Fluor 488 goat anti-rabbit IgG, 1:400; Alexa Fluor 488 Donkey anti-Goat IgG, 1:400; Alexa Fluor 594 goat anti-mouse IgG, 1:400; and Alexa Fluor 594 goat anti-rabbit IgG, 1:400). All secondary antibodies were purchased from Invitrogen. Phase-contrast images were acquired on a Zeiss LSM 880 inverted Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany).
5
Myocardial Infarction Tissue Analysis
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Rats were sacrificed 4 weeks after myocardial infarction. For immunohistochemistry, hearts were embedded in paraffin and cut into 8-mm thin sections. The tissues were stained by Masson trichrome staining (HT15 Trichrome Stain Kit, Sigma). Viable tissue and scar area within the infarcted region were measured on each section by tracing the infarcted borders manually using the ImageJ software. Six sections were analyzed per heart.
SD model rats were anesthetized with chloral hydrate at a dose of 20 mg/kg, intubated, and ventilated 4 weeks after myocardial infarction. SD rats were treated with intravenous injection of 10 units heparin, followed by injection of 200 µl staining reagent Griffonia Simplicifolia lectin I (Vector Labs, CA USA). Rats were kept in a ventilator for 10 min, and the heart tissues were fixed with 10% paraformaldehyde. The new functional capillary density of hearts was measured by anti-Griffonia simplicifolia I (lectin 1:100) and anti-Alexa Fluor 594 donkey anti-goat IgG antibodies. Alpha-smooth muscle actin (α-SMA) is commonly used as a marker of myofibroblast formation (BioLegend, CA, USA). Newly formed blood vessels were also counted as α-SMA under the fluorescence microscope. Inflammation in myocardial infarction tissue was observed by hematoxylin-eosin staining.
SD model rats were anesthetized with chloral hydrate at a dose of 20 mg/kg, intubated, and ventilated 4 weeks after myocardial infarction. SD rats were treated with intravenous injection of 10 units heparin, followed by injection of 200 µl staining reagent Griffonia Simplicifolia lectin I (Vector Labs, CA USA). Rats were kept in a ventilator for 10 min, and the heart tissues were fixed with 10% paraformaldehyde. The new functional capillary density of hearts was measured by anti-Griffonia simplicifolia I (lectin 1:100) and anti-Alexa Fluor 594 donkey anti-goat IgG antibodies. Alpha-smooth muscle actin (α-SMA) is commonly used as a marker of myofibroblast formation (BioLegend, CA, USA). Newly formed blood vessels were also counted as α-SMA under the fluorescence microscope. Inflammation in myocardial infarction tissue was observed by hematoxylin-eosin staining.
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