Hearts of day 2 and day 7 post-MI were dissected to collect infarct, border-zone, and remote myocardium, which was snap frozen in liquid nitrogen for protein and RNA analysis. Hearts at day 7 and day 30 post-MI were fixed in 10% neutral buffered formalin for histology. The mid-myocardium was cut into 5-μm sections and stained with Masson trichrome or picro-sirius red (Sigma-Aldrich, Singapore) plus immunofluorescence staining for α-SMA (clone 1A4; Sigma-Aldrich, Singapore) and DAPI (Thermo Fisher Scientific, Singapore). Bright-field and fluorescent images were captured (Nikon Eclipse Ti inverted microscope with C-HGFIE pre-centered fiber illuminator, Nikon, Singapore) and analyzed using ImageJ 1.45 s.37 (link)
α sma clone 1a4
Manufactured by Merck Group
Sourced in United States, Germany
α-SMA (clone 1A4) is a mouse monoclonal antibody that recognizes the alpha-smooth muscle actin isoform. It is commonly used as a marker for the identification of smooth muscle cells and myofibroblasts in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated polyclonal goat anti rat antibody, by Thermo Fisher Scientific (2 mentions)
Cy3 conjugated mouse monoclonal anti α smooth muscle actin antibody, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Masson trichrome, by Merck Group (1 mentions)
Picrosirius red, by Merck Group (1 mentions)
C hgfie pre centered fiber illuminator, by Nikon (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Bleomycin, by Teva Pharmaceuticals (1 mentions)
Fibronectin, by Abcam (1 mentions)
Phosphor smad3, by Cell Signaling Technology (1 mentions)
Actinomycin d act d, by MedChemExpress (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Actin, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
Dab substrate, by Thermo Fisher Scientific (1 mentions)
Clone 7h8.2c12, by BD (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
15 protocols using α sma clone 1a4
1
Myocardial Infarction Tissue Analysis
2
TGF-β1-Induced Fibrosis Signaling
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Recombinant human transforming growth factor-β1 (TGF-β, 7754-BH-025/CF) was purchased from R&D Systems, Inc. (Minneapolis, MN). Bleomycin (15 Unit/vial, Teva) was purchased from the Pharmacy of the University of Texas Health Science Center at Tyler. Small molecular inhibitors including SIS3 (HY-13013), SB230580 (HY-10256), ICG001 (HY-14428) and Actinomycin D (Act D, HY-17559) were purchased from MedChemExpress (Monmouth Junction, NJ). The primary antibodies used in the study includes PD-L1 (clone E1L3N and 405.9A11, Cell Signaling Technology), Smad3 (clone 38-Q, Santa Cruz Biotechnology), phosphor-Smad3 (9520S, Cell Signaling Technology), α-SMA (clone 1A4, Sigma), Collagen type 1 (1310-08, SouthernBiotech)16 (link), Fibronectin (ab2413, Abcam), phosphor-GSK3α/β (Ser21/Ser9, 9331S, Cell Signaling Technology), GSK3β (12456S, Cell Signaling Technology), β-catenin (Cat#sc-7963, Santa Cruz), Actin (A2066, Sigma), and GAPDH (1E6D9, Proteintech).
3
Histological Analysis of Liver Specimens
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Preparation and staining of liver specimens were performed as previously reported [16 (link)]. Briefly, liver specimens were fixed in 10% formalin and embedded in paraffin. Then the sections were stained in 0.1% Sirius Red F3BA in a saturated picric acid solution. Randomly selected five fields from each section were photographed and analyzed. Red staining areas were quantified using NIH ImageJ software and expressed as a percentage of the total analyzed areas.
For immunohistochemical staining, primary antibodies for α-SMA (clone 1A4; Sigma-Aldrich), CD45 (PA5-87,427, Invitrogen) were incubated, followed by incubation of species-matched secondary antibody labelled with HRP (Thermo Scientific, USA) and detection with DAB substrate (Thermo Scientific). The nuclei were counterstained with hematoxylin.
For immunofluorescence, cells were cultured on BD Falcon™ 8-well Culture Slides, fixed and permeabilized, incubated with anti-alpha-SMA antibody and species-matched secondary antibody conjugated with Alexa Fluor-594. The nuclei were stained with DAPI (Sigma). Cell apoptosis was analyzed using a TdT-mediated dUTP nick end labeling (TUNEL) kit (R&D Systems, Inc.) according to the manufacturer’s instructions.
For immunohistochemical staining, primary antibodies for α-SMA (clone 1A4; Sigma-Aldrich), CD45 (PA5-87,427, Invitrogen) were incubated, followed by incubation of species-matched secondary antibody labelled with HRP (Thermo Scientific, USA) and detection with DAB substrate (Thermo Scientific). The nuclei were counterstained with hematoxylin.
For immunofluorescence, cells were cultured on BD Falcon™ 8-well Culture Slides, fixed and permeabilized, incubated with anti-alpha-SMA antibody and species-matched secondary antibody conjugated with Alexa Fluor-594. The nuclei were stained with DAPI (Sigma). Cell apoptosis was analyzed using a TdT-mediated dUTP nick end labeling (TUNEL) kit (R&D Systems, Inc.) according to the manufacturer’s instructions.
4
Multicolor Immunofluorescence Staining of Tumor Vasculature
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ECs were stained with rat monoclonal anti-mouse CD31 antibody (PEACAM-1; clone MEC 13.3, 1:500; BD Pharmingen, Heidelberg, Germany) and detected using an Alexa Fluor 488-conjugated polyclonal goat anti-rat antibody (1:500; Invitrogen). Proliferating ECs in tumor tissue were stained with a rat anti-mouse CD105 (endoglin) PE-conjugated monoclonal antibody (1:500, clone: MJ7/18; eBioscience, Frankfurt am Main, Germany). Perivascular cells were stained with Cy3-conjugated mouse monoclonal anti-α-smooth muscle actin antibody (1:1,000, α-SMA clone 1A4; Sigma-Aldrich, Munich, Germany). Mitochondrial morphology and membrane potential were visualized by MitoTracker (RedCMXRos) staining (Invitrogen). For cytochrome c staining, a mouse monoclonal anti-cytochrome c antibody was used (1:500; clone 7H8.2C12; BD Biosciences) and detected with a secondary Alexa Fluor 488-conjugated polyclonal goat anti-mouse antibody (1:500; Invitrogen). For VE-cadherin staining, a monoclonal rabbit anti-VE-cadherin antibody (clone D87F2; Cell Signaling) was used and detected with a secondary Alexa Fluor 488-conjugated polyclonal goat anti-rabbit antibody (1:500; Invitrogen).
5
Kidney Tissue Protein Extraction and Analysis
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Total proteins were extracted from kidney tissue samples when treated with radioimmunoprecipitation assay (RIPA) lysis (Sangon Biotech, Shanghai, China). The concentrations of total proteins were measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred to an NC membrane. The NC membrane was blocked with 5 % non-fat milk and then incubated with primary antibodies against α-SMA (clone 1A4, 1:1000, A2547, Sigma-Aldrich, St. Louis, MO, USA) and fibronectin (clone 3E2, 1:1000, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C, respectively. Then, the membrane was washed with TBST and incubated with secondary antibodies (1:5000) at 37 °C for 2h. Finally, the proteins were monitored with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, NJ, USA) and then quantitatively analyzed by ImageJ software (version 1.4.0., National Institutes of Health). GAPDH protein was used for the internal control.
6
Immunostaining of Tumor Immune Cells
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Immunostaining of immune cells from B16F0 tumour sections was performed as described previously (Reynolds et al., 2010 (link)).
Primary mouse lung fibroblasts and endothelial cells were fixed with 4% PFA for 10 min, washed twice and blocked with 5% NGS in PBS for 30 min at room temperature. Primary antibodies to vimentin (1:100; 5741, Cell Signaling), NG2 (1:100; MAB 5385, Millipore), endomucin (1:100; V7C7, Santa Cruz Biotechnology) and α-Sma (clone 1A4, Sigma) were incubated for 1 h at room temperature, washed three times with PBS, and incubated with the relevant secondary antibody for 45 min at room temperature.
Primary mouse lung fibroblasts and endothelial cells were fixed with 4% PFA for 10 min, washed twice and blocked with 5% NGS in PBS for 30 min at room temperature. Primary antibodies to vimentin (1:100; 5741, Cell Signaling), NG2 (1:100; MAB 5385, Millipore), endomucin (1:100; V7C7, Santa Cruz Biotechnology) and α-Sma (clone 1A4, Sigma) were incubated for 1 h at room temperature, washed three times with PBS, and incubated with the relevant secondary antibody for 45 min at room temperature.
7
Cell Culture and Immunostaining Protocol
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Fetal bovine serum (FBS), Dulbecco’s
modified Eagle’s medium (DMEM), Trypsin-EDTA, CCK-8, DBCO-Cy3,
Triton X-100, TGFβ1, 4′,6-diamidino-2-phenylindole (DAPI),
and primary antibodies against human alpha smooth muscle actin (α-SMA,
clone 1A4) and produced in mice were purchased from Sigma (St. Louis,
MO, USA). Primary rabbit anti-human collagen type 1 (COL-1) was bought
from Cedarlane Laboratories (Burlington, Canada). Phalloidin Alexa
Fluor 568, Calcein Am, and TOTO-3 were purchased from Invitrogen.
Alexa Fluor 488 labeled goat anti-mouse secondary antibody was obtained
from Molecular Probes Life Technologies. Alexa Fluor 488 labeled goat
anti-rabbit secondary antibody was from Invitrogen (Thermo Fisher
Scientific, United Kingdom). CNA35-OG488 was obtained from the Department
of Biomedical Engineering (TU/e Eindhoven, The Netherlands). For purified
water, Milli-Q was used.
modified Eagle’s medium (DMEM), Trypsin-EDTA, CCK-8, DBCO-Cy3,
Triton X-100, TGFβ1, 4′,6-diamidino-2-phenylindole (DAPI),
and primary antibodies against human alpha smooth muscle actin (α-SMA,
clone 1A4) and produced in mice were purchased from Sigma (St. Louis,
MO, USA). Primary rabbit anti-human collagen type 1 (COL-1) was bought
from Cedarlane Laboratories (Burlington, Canada). Phalloidin Alexa
Fluor 568, Calcein Am, and TOTO-3 were purchased from Invitrogen.
Alexa Fluor 488 labeled goat anti-mouse secondary antibody was obtained
from Molecular Probes Life Technologies. Alexa Fluor 488 labeled goat
anti-rabbit secondary antibody was from Invitrogen (Thermo Fisher
Scientific, United Kingdom). CNA35-OG488 was obtained from the Department
of Biomedical Engineering (TU/e Eindhoven, The Netherlands). For purified
water, Milli-Q was used.
8
Quantifying Cellular Markers in Tissue Sections
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Primary antibodies were anti–Gal-1 (AF1152, R&D Systems), CD68 (Ab53444, Abcam), and α-SMA (clone 1A4, Sigma-Aldrich). Rabbit anti-goat horseradish peroxidase (HRP), donkey anti-rat HRP (The Jackson Laboratory), and Alexa Fluor 488 donkey anti-rat (Invitrogen) were used as secondary antibodies. HRP was then added, and sections were stained with 3,3′-diaminobenzidine (DAB) substrate-chromogen (DAKO) and counterstained with hematoxylin. Computer-assisted morphometric analysis was performed with Image-Pro Plus software (version 1.0 for Windows). The threshold setting for area measurement was equal for all images. Samples from each animal were examined in a blinded manner. Results were expressed as % positive area versus total area (CD68 and α-SMA).
9
Fibrosis Biomarker Characterization Protocol
10
Fibroblast Identification via Immunofluorescence
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Fibroblasts were identified using immunofluorescence staining. Cells were seeded 1.0 ×
105 on poly-L-lysine-coated coverslips for 24-well plates (Matsunami Glass,
Osaka, Japan), cultured for 48–72 hr, and washed by phosphate-buffered saline (PBS; Wako).
The cells were fixed with paraformaldehyde at room temperature for 10 min. After fixation,
the cells were permeabilized with 0.5% Triton X-100 (Wako) in PBS, blocked with 10% goat
serum albumin (Wako) for 30 min, and immunostained overnight at 4°C with primary mouse
antibodies for vimentin (a marker for mesenchymal cells) (clone V9, Dako, Tokyo, Japan),
cytokeratin (a marker for epithelial cells) (clone AE1/AE3, Dako), and α-SMA (clone 1A4,
Sigma Aldrich) diluted 1:2,000 in PBS. The cells were incubated at room temperature for 1
hr with fluorochrome-conjugated secondary goat anti-mouse antibody (Alexa-Fluor-488,
catalog number A21202, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS,
and nuclei phenylindole (DAPI; Dojindo, Kumamoto, Japan) at room temperature for 15 min.
The coverslips were mounted on glass slides. Images were obtained using a confocal
microscope (LSM700; Zeiss, Oberkochen, Germany).
105 on poly-L-lysine-coated coverslips for 24-well plates (Matsunami Glass,
Osaka, Japan), cultured for 48–72 hr, and washed by phosphate-buffered saline (PBS; Wako).
The cells were fixed with paraformaldehyde at room temperature for 10 min. After fixation,
the cells were permeabilized with 0.5% Triton X-100 (Wako) in PBS, blocked with 10% goat
serum albumin (Wako) for 30 min, and immunostained overnight at 4°C with primary mouse
antibodies for vimentin (a marker for mesenchymal cells) (clone V9, Dako, Tokyo, Japan),
cytokeratin (a marker for epithelial cells) (clone AE1/AE3, Dako), and α-SMA (clone 1A4,
Sigma Aldrich) diluted 1:2,000 in PBS. The cells were incubated at room temperature for 1
hr with fluorochrome-conjugated secondary goat anti-mouse antibody (Alexa-Fluor-488,
catalog number A21202, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS,
and nuclei phenylindole (DAPI; Dojindo, Kumamoto, Japan) at room temperature for 15 min.
The coverslips were mounted on glass slides. Images were obtained using a confocal
microscope (LSM700; Zeiss, Oberkochen, Germany).
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