For the imaging of constructed neural networks and vascular networks, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min. After permeabilization, the cells were treated with BlockAce (Dainippon Pharmaceutical, Japan) for 1 hour to block non-specific staining. The cells were then incubated at 4 °C overnight with primary antibodies, a mouse anti-α-SMA antibody (Sigma-Aldrich) for pericytes, a sheep anti-PECAM-1 antibody for BMECs, a mouse anti-Tuj-1 or anti-MAP2 antibodies for neurons. Thereafter, the cells were treated with secondary antibodies, Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen), Alexa Fluor 594-conjugated anti-rabbit IgG (Invitrogen) and Alexa Fluor 647-conjugated anti-rabbit IgG (Invitrogen), and incubated at room temperature for 2 hours. The cells were finally incubated with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for staining cell nucleus. The cells were rinsed with PBS between each step. Z-stack fluorescent images were taken by a confocal laser-scanning microscope (LSM700, Carl Zeiss, Germany). The 2D projection images were generated with the z-stack fluorescent images using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Mouse anti α sma antibody
Manufactured by Merck Group
Sourced in Germany, China
The Mouse anti-α-SMA antibody is a laboratory reagent used for the detection and quantification of alpha-smooth muscle actin (α-SMA) in biological samples. It is a mouse monoclonal antibody that specifically binds to α-SMA, a structural protein found in smooth muscle cells. This antibody can be used in various immunoassay techniques, such as Western blotting and immunohistochemistry, to identify and analyze the presence and distribution of α-SMA in research or diagnostic applications.
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Lab products found in correlation
Biotinylated donkey anti rat secondary antibody, by Abcam (2 mentions)
Histoquant, by 3DHISTECH (2 mentions)
Mouse anti vinculin antibody, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti fibronectin antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd68 antibody, by Abcam (1 mentions)
Goat anti type 3 collagen antibody, by Southern Biotech (1 mentions)
Goat anti vimentin antibody, by Santa Cruz Biotechnology (1 mentions)
F0250, by Agilent Technologies (1 mentions)
F0205, by Agilent Technologies (1 mentions)
V9131, by Merck Group (1 mentions)
14 protocols using mouse anti α sma antibody
1
Immunofluorescence Imaging of Neural and Vascular Networks
2
Hepatic alpha-SMA Immunohistochemistry
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Immunohistochemical stainings for hepatic αSMA were prepared using a cryostat. Cryo sections (4–6μm) of snap-frozen liver samples were fixed and incubated with mouse-anti-αSMA antibody (Sigma Aldrich, München, Germany). Thereafter, a biotinylated donkey-anti-rat secondary antibody was used (Abcam, Cambridge, UK). Sections were detected and quantified using computerized image capture device (Histoquant; 3DHistech, Budapest, Hungary). Results are expressed as mean±SEM.
3
Immunofluorescence Analysis of Fibrotic Markers
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Recombinant human follistatin was provided by Dr. Y. Eto (Central Research Laboratory, Ajinomoto, Kawasaki, Japan). Antibodies used in this study were as follows: goat anti-type I collagen antibody (1 : 100), goat anti-type III collagen antibody (1 : 100) (Southern BioTech, Birmingham, AL), mouse anti-α-SMA antibody (1 : 100) (Sigma, St. Louis, MO), mouse anti-CD68 antibody (1 : 100) (Abcam, Cambridge, UK), rabbit anti-inhibin βA antibody (1 : 100) (Thermo Fisher Scientific, Yokohama, Japan), rabbit anti-fibronectin antibody (1 : 100), goat anti-vimentin antibody (1 : 100) (Santa Cruz biotechnology, Inc., CA), rabbit anti-CD3 antibody (1 : 100) (Vector Labs, Burlingame, CA), Alexa Fluor 488 goat anti-mouse IgG (1 : 2000), and Alexa Fluor 488 goat anti-rabbit IgG (1 : 2000) (Invitrogen, Carlsbad, CA).
4
Immunohistochemical Analysis of RAGE, S100A8, and S100A9 in PAH
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Immunohistochemical and immunocytochemical analyses were performed to confirm the expression of RAGE, S100A8 and S100A9 in sections of lungs and in PASMCs. For immunohistochemical analysis, lungs from patients with PAH or control subjects were fixed with 4% paraformaldehyde. Immunofluorescence was performed on 5-μm lung slices. For immunocytochemical analysis, PASMCs were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Rabbit anti-human RAGE antibody (Santa Cruz Biotechnology), mouse anti-αSMA antibody (Sigma-Aldrich), goat anti-S100A8 (calgranulin A) antibody (Santa Cruz Biotechnology) and goat anti-S100A9 (calgranulin B) antibody (Santa Cruz Biotechnology) were used. The second antibodies were swine anti-rabbit (FITC, F0205) (Dako), rabbit anti-mouse (TRITC, R0270) (Dako) and rabbit, anti-goat (FITC, F0250) (Dako).
5
Visualizing Cell Adhesion and Differentiation
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All samples were washed with PBS to remove any unattached cells and fixed in situ with 4% paraformaldehyde for 10 min at room temperature. Then, cells were permeabilized for 5 min with PBS containing 0.1% TritonX-100 and unspecific binding blocked with 2% BSA in PBS for 1 h. To visualize focal adhesions (FAs) or differentiation, cells were treated with the three primary antibodies, including mouse anti-vinculin antibody (Sigma; V9131; 1:300), mouse anti-types I collagen (Col-I) antibody (Abcam; ab6308; 1:200), or mouse anti-α-SMA antibody (Sigma; A5228; 1:200) at 4°C overnight, followed by incubation with goat anti-mouse-FITC (Sigma; F0257; 1:100) at 37°C for 1 h. Subsequently, F-actin was stained with phalloidin-TRITC (Cytoskeleton; PHDR1; 100nM) and the nucleus with DAPI (Thermo; P36931; undiluted).
All specimens were then examined under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at low magnification (5× or 10×) or a laser scanning confocal microscopy (Nikon Co., Japan) at high magnification (40×). The fluorescent images were quantitatively analyzed using NIH ImageJ software.
All specimens were then examined under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at low magnification (5× or 10×) or a laser scanning confocal microscopy (Nikon Co., Japan) at high magnification (40×). The fluorescent images were quantitatively analyzed using NIH ImageJ software.
6
Western Blot Analysis of Liver Tissues
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The protein extracts from liver tissues and CFSC cells were prepared in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktails (Roche, Shanghai, China). Then, the protein samples were separated using SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Shanghai, China). After blocking with 5% skim milk, the membranes were incubated with mouse anti-α-SMA antibody (Sigma Aldrich, Shanghai, China) or rabbit anti-GAPDH antibody (Proteintech, Wuhan, China) overnight at 4 °C and subsequently incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China) for 2 h at room temperature. Finally, the bands were examined with an ECL assay kit (NCM Biotech, Suzhou, China).
7
Immunofluorescent Analysis of Pulmonary Artery Remodeling
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Lung tissues were fixed in formalin and embedded in paraffin (Leica), and 6 nm sections of lung tissues were deparaffinized and permeabilized with xylol and then incubated with a mouse anti-α-SMA antibody, goat anti-mouse IgG, and DAPI. Briefly, the sections were blocked with PBS containing 5% Tween and 5% bovine serum albumin (Sangon Biotech Corp.) for 45 min at room temperature. Then, the sections were incubated with a mouse anti-α-SMA antibody (1:1,000; Sigma) overnight at 4°C and washed three times with PBST. Subsequently, the sections were incubated with FITC-conjugated goat anti-mouse IgG (1:500; Invitrogen Life Technologies) for 60 min and washed three times with PBST. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and washed three times with PBS. A confocal microscope system was used to examine pulmonary artery (PA) remodeling (Zeiss LSM-710, Germany). Images were acquired using ZEN2.3 software.
8
Western Blot Analysis of Protein Expression
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Total cellular protein was extracted with RIPA lysis buffer, and protein concentration of lysates was measured by BCA Protein Assay Kit (Thermo, Waltham, USA). The protein samples were stored at −80°C for further analysis. Subsequently, equal amounts of proteins were separated by 10% SDS-PAGE gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). After blocking in 5% non-fat milk in TBST buffer for 1 h at room temperature, the membranes were incubated with three primary antibodies, respectively, including mouse anti-vinculin antibody (Sigma; V9131; 1:1500), mouse anti-Col-I antibody (Abcam; ab88147; 1:2000), mouse anti-α-SMA antibody (Sigma; A5228; 1:2000), or mouse anti-β-actin antibody (Affinity; T0022; 1:3000) at 4°C overnight, followed by incubation with HRP-linked goat anti-mouse IgG (H + L) (Affinity; S0002; 1:3000) at 37°C for 1 h. After washing the membranes with TBST, the band signals were visualized by using Affinity® ECL Reagent. Finally, the membranes were exposed and analyzed by using Luminescent Image Analyzer (Amersham, Uppsala, Sweden). The results of each blot were normalized against β-actin protein expression.
9
Immunofluorescence Analysis of Fibrosis Markers
10
Immunohistochemical Analysis of Aortic CD68 and α-SMA
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For immunohistochemistry, dewaxed aortic root sections were fixed with cold acetone for 10 min, then incubated with mouse anti-CD68 (1:100, Abcam) at 4°C overnight. After washing three times with PBS, sections were incubated with goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, United States) at 37°C for at least 1 h. Protein expression was visualized using 3,3′-diaminobenzidine (Vector Laboratories, CA) for 1.5 min, and hematoxylin was used to stain the nuclei.
For immunohistochemistry staining of aortic sections by anti-CD68 and α-smooth muscle actin (α-SMA), dewaxed aortic sections were boiled in 10 mM citrate (pH 6.0) for antigen retrieval, then incubated with mouse anti-CD68 (1:100, Abcam, Ab31630) and mouse anti-α-SMA antibody (1:200, Sigma, A2547) at 4°C overnight. After washing three times with PBS, sections were incubated with goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, United States) at 37°C for at least 1 h. Protein expression was visualized using 3,3′-diaminobenzidine (Vector laboratories, CA) for 1.5 min, and hematoxylin was used to stain the nuclei.
For immunohistochemistry staining of aortic sections by anti-CD68 and α-smooth muscle actin (α-SMA), dewaxed aortic sections were boiled in 10 mM citrate (pH 6.0) for antigen retrieval, then incubated with mouse anti-CD68 (1:100, Abcam, Ab31630) and mouse anti-α-SMA antibody (1:200, Sigma, A2547) at 4°C overnight. After washing three times with PBS, sections were incubated with goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, United States) at 37°C for at least 1 h. Protein expression was visualized using 3,3′-diaminobenzidine (Vector laboratories, CA) for 1.5 min, and hematoxylin was used to stain the nuclei.
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