Tissues were fixed in 4% PFA, embedded in paraffin, and sectioned at 4–7 µm thickness. Immunostainings were performed using the following antibodies: α-SMA (1:500, Sigma-Aldrich, Cat. no A5228), CD31 (1:50, Dako, Cat. no M0823), VEGFR-1 (1:250, Santa Cruz Biotechnology, Cat no sc-31173), VEGFR-2 (1:250, Santa Cruz Biotechnology, Cat. no sc-6251) and VEGF-A (1:500, Santa Cruz Biotechnology, Cat no sc-7269). Photographs were taken with Olympus AX70 microscope (Olympus Optical, Tokyo, Japan). For whole-mount tissue imaging, 1 mm thick longitudinal sections were cut and stained with CD31 (1:100, Dako, Cat. no M0823) and α-SMA conjugated with Cy3 (1:500, Sigma-Aldrich, Cat. no C6198). For CD31, goat anti-mouse Alexa 488 (1:500; Invitrogen, Cat. no A-11001) was used. Imaging was performed by Nikon A1R multiphoton microscope (MPLSM) and LSM700 Zeiss confocal microscope (LSM).
α sma
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Macao, China, Sao Tome and Principe, Japan, Italy, Israel
α-SMA is a common marker for the identification of activated myofibroblasts. It is a contractile protein that is expressed in smooth muscle cells and myofibroblasts. α-SMA is often used in research applications to study fibrosis and tissue remodeling.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (29 mentions)
Gapdh, by Cell Signaling Technology (20 mentions)
E cadherin, by BD (17 mentions)
F4 80, by Bio-Rad (16 mentions)
β actin, by Santa Cruz Biotechnology (14 mentions)
Gapdh, by Santa Cruz Biotechnology (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (13 mentions)
Vimentin, by Cell Signaling Technology (13 mentions)
Cleaved caspase 3, by Cell Signaling Technology (13 mentions)
Collagen 1, by Abcam (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions)
P smad3, by Cell Signaling Technology (12 mentions)
Fibronectin, by Abcam (12 mentions)
Hoechst 33342, by Thermo Fisher Scientific (12 mentions)
Fibronectin, by Santa Cruz Biotechnology (11 mentions)
α tubulin, by Merck Group (11 mentions)
Gapdh, by Merck Group (11 mentions)
Collagen 1, by Southern Biotech (10 mentions)
Smad3, by Cell Signaling Technology (10 mentions)
Vimentin, by Merck Group (10 mentions)
501 protocols using α sma
1
Immunohistochemical Tissue Analysis
2
Immunofluorescence Staining of Colon Tissues
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FFPE colon sections were probed with antibodies against F4/80 (AbD Serotec), Gr-1 (AbD Serotec), CD3 (Abcam), B220 (BD), p-STAT3 (Cell Signaling Technology), Vimentin (Abcam), and α-SMA (FITC-conjugated; Sigma-Aldrich). The anti–rabbit Alexa Fluor 568–conjugated secondary antibody (Invitrogen), biotinylated secondary antibodies (Vector Laboratories), the Vectastain ABC kit (Vector Laboratories), and the Vectastain DAB kit or the Vectastain NovaRED kit (Vector Laboratories), were used for signal amplification and detection. Images were acquired with an Eclipse E800 microscope (Nikon) equipped with a Dxm1200F camera (Nikon). Cryosections of paraformaldehyde (PFA)-fixed tissue samples or PFA-fixed cells were probed with antibodies against Vimentin (Abcam), α-SMA (Sigma-Aldrich), and Collagen IV (Abcam), followed by secondary antibodies conjugated with Alexa Fluor 647 (Invitrogen). Images were acquired with a TCS SP8X White Light Laser confocal system (Leica).
3
Western Blot Analysis of Fibrosis Markers
4
Histological Analysis of Liver Sections
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For histological analysis, liver sections were stained with Hematoxylin-eosin (H&E). The primary antibodies applied to the tissue sections were a rat antibody against F4/80 (dilution 1:500; Serotec, Oxford, UK), a mouse antibody against α-SMA (1:200), 4-HNE (1:200, Millipore, Billerica, MA, USA), CYP2E1 (1:200), and Prx1 and Prx6 (1:200). Peroxidase activity was revealed using DAB substrate, slides were counterstained with Hematoxylin (Sigma Chemical, St. Louis, MO, USA), then dehydrated and mounted in Safemount embedding medium (Labonord, France).
5
Investigating Thymosin β4 Modulation of Inflammation
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Thymosin β4 (HPLC > 98%) was purchased from GL Biochem. (Shanghai) Ltd and dissolved in 0.9% saline. α-SMA (Cat: CBL171) and β-actin (Cat: MAB1501), nitro-tyrosine (N-Tyr) (Cat: 05–233) antibodies were bought from Millipore (Darmstadt, Germany). P65 (Cat: 8242) antibody was purchased from Cell Signaling Technology (Denver, Colorado, USA). TGF-β1 (Cat: 18978-1-AP) antibody was bought from Proteintech (Wuhan, China). TNF-α (Cat: ab6671) and IL-1β (Cat: ab2105) antibodies were purchased from Abcam (Shanghai, China). Secondary antibodies against rabbit (Cat: 111-035-003) and mouse (Cat: 115-035-003) were obtained from Jackson ImmunoResearch (Baltimore, Maryland, USA). Immobilon enhanced chemiluminescence (ECL) detection reagent (Cat: WBKLS0500) was purchased from Millipore (Darmstadt, Germany). All other reagents were of analytic grade.
6
Immunofluorescence Staining of Tissue Sections
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Tissue sections that were embedded in paraffin slides were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The endogenous peroxidase activity was inhibited by incubating the slide in 3% (v/v) hydrogen peroxide, and the antigenicity was recovered with a 0.01 mol l−1 sodium citrate buffer (pH 6.0, Cat. No. AR0024, Boster Biological Technology, Wuhan, China). The samples were incubated overnight at 4°C with antibodies for UCHL1 (dilution: 1:100, Cat. No. 13179, Cell Signaling Technology, Danvers, MA, USA), SOX9 (dilution: 1:100, Cat. No. AB5535, Millipore, Burlington, MA, USA), and α-SMA (dilution: 1:100, Cat. No. ARG52485, Arigo, Hsinchu, Taiwan, China); washed with PBS; and then incubated with Cy3-conjugated goat anti-rabbit (dilution: 1:100, Cat. No. CW0159S, CWBIO, Beijing, China), Cy3-conjugated goat anti-mouse (dilution: 1:100, Cat. No. CW0145, CWBIO), and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (dilution: 1:100, Cat. No. CW0114S, CWBIO) at room temperature for 30 min. Subsequently, the sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 10 μg ml−1, Cat. No. C0065, Solarbio, Beijing, China). Immunofluorescence signals were detected by fluorescence microscopy (IX71, OLYMPUS).
7
Immunocytochemical Analysis of Protein Expression
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To evaluate protein expression and localization, immunocytochemical staining was performed. Before using the cell culture slides, they were coated with poly-L-lysine for 4 h, after which they were exposed to UV light for 6 h. Cells were seeded on the poly-L-lysine coated slides. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with phosphate-buffered saline containing 0.01% Triton X-100 for 10 min. Next, the cells were blocked with 3% bovine serum albumin for 1 h, incubated with primary antibodies; α-SMA (Millipore), Fibronectin (Santa Cruz Biotechnology, Inc), and collagen type 1 (Abcam), followed by incubation with secondary antibodies (Invitrogen). Finally, the cells were counterstained with 4’-6-diamidino-2-phenylindole for 10 min and the stained cells were visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).
8
Nasal Fibroblast Protein Expression Analysis
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Nasal fibroblasts were seeded into a 60 mm culture dish with 5×105 cells/mL and lysed in Ripa buffer (Sigma-Aldrich) with protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore Inc., Billerica, MA). Membranes were blocked with a 5% bovine serum albumin. The blots were incubated with primary antibodies against α-SMA (Millipore Inc.), fibronectin (Santa Cruz Biotecknology Inc., CA ), collagen type I (Abcam, Cambridge, UK), phospho-p38, total-p38, phospho-JNK, total-JNK, phospho-ERK, total-ERK (Cell Signaling, MA), total-p50, phospho-p50 (Santa Cruz Biotecknology Inc.), and β-actin (Santa Cruz Biotecknology Inc.) overnight at 4°C. HRP-conjugated secondary antibodies was incubated for 1 hour at room temperature and detected with Amersham imager 600 (GE healthcare, little Chalfont, UK)
9
Dihydrorotenone-Mediated Cellular Assays
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Dihydrorotenone was provided by Dr. Kyeong Kyu Kim at Sungkyunkwan University (Suwon, Korea). An annexin-V staining kit was purchased from BD Bioscience (Franklin Lakes, NJ). An EZ-cytox cell viability assay kit was purchased from Daeill Lab (Seoul, Korea). A cell contraction assay kit was purchased from Cell Biolabs, Inc (San Diego, CA). Twist1 (Cat: ab50887, Abcam, Cambridge, MA), FSP1 (Cat: 07-2274, Millipore, Burlington, MA), PDGFRα (Cat: S3164, Cell Signaling, Danvers, MA), PDGFRβ (Cat: ab32570, Abcam, Cambridge, MA), FAPα (Cat: 53066, Abcam, Cambridge, MA), α-SMA (Cat: (E184) 04-1094, Millipore, Burlington, MA) and α-tubulin antibodies (Cat: SC-8035, Santa Cruz, Dallas, TX) were used for immunoblotting.
10
Liver Protein Extraction and Western Blot
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The liver tissue samples were homogenized in lysis buffer (1% Triton X-100, 20 mM Tris, 40 mM NaF, 0.2% SDS, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 100 mM NaCl, pH 7.5) and then centrifuged for 15 minutes at 15,000× g to obtain the liver homogenates. β-actin (anti-β-actin polyclonal antibodies, ABT264, Millipore, Temecula, CA, USA) and α-SMA (rabbit anti-αSMA monoclonal antibodies, Millipore, Temecula, CA, USA) antibodies were used for Western blotting.
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