The sectioned kidney tissues were incubated with lysis buffer (iNtRON Biotechnology Inc., Seongnam, Korea, http://www.intronbio.com ) on ice for 20 min. Collected lysates were boiled for 5 min and separated on a 9% gradient acrylamide gel and then transferred electrophoretically to polyvinyl difluoride membranes (MilliporeSigma, Burlington, MA, https://www.sigmaaldrich.com ). Nonspecific binding of antibody was blocked with 5% bovine serum albumin. Immunodetection was performed with to an anti-α smooth muscle actin (α-SMA) primary antibody (1 : 2,000; Abcam, Cambridge, U.K., http://www.abcam.com ) followed by incubation with the peroxidase-conjugated secondary antibody (1 : 5,000; Enzo Life Sciences, Farmingdale, NY, https://www.enzolifesciences.com ), and the blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). Primary and secondary antibodies were diluted in 5% skim milk (bioWORLD, Dublin, OH, http://www.bio-world.com ) in PBS. All blots were normalized against α-tubulin to control for protein loading. The immunoblot bands were detected using Immobilon™ Western HRP substrate peroxide Solution (Thermo Fisher Scientific Inc.).
α sma primary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The α-SMA primary antibody is a tool used in research applications. It is designed to detect the presence of the alpha-smooth muscle actin (α-SMA) protein, which is a key marker of myofibroblasts and is involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Skim milk, by Bioworld Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Enzo Life Sciences (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by iNtRON Biotechnology (1 mentions)
P0260, by Beyotime (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Odyssey ir imager, by LI COR (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
Hrp conjugated goat anti rat igg, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Optical microscope, by Olympus (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Normal goat serum (ngs), by Beyotime (1 mentions)
Duoset elisa, by R&D Systems (1 mentions)
16 protocols using α sma primary antibody
1
Western Blot Analysis of Renal α-SMA
2
Immunocytochemistry of Collagen Gel
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Immunocytochemistry was performed on collagen gel by first fixing and permeabilized using cold methanol for 5 min at − 20 °C. Following a brief PBS wash, 1% BSA was used to block non-specific staining for 1 h at room temperature. Then, gels were incubated with α-SMA primary antibody overnight (Abcam, 1:100) at 4 °C. The following day, collagen gels were washed in PBS and incubated with a secondary antibody (Anti-rabbit Alexa Fluor 488) for 1 h. Finally, gels were stained with DAPI containing antifade solution and visualized using a confocal scanning electron microscope (Carl-Zeiss).
3
Immunofluorescence Assay for α-SMA
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The FLS were stimulated by TGF-β1 and treated with LIPUS, then fixed with 4 % PFA for 20 min. After washing three times with PBS, cells were permeabilized with 0.01 % Triton X-100 in PBS for 20 min, and then blocked for 1 h with blocking buffer (P0260, Beyotime). Cells were incubated with α-SMA primary antibody (1:200 dilution; ab32575, Abcam, USA) overnight at 4 °C. Cells were washed three times with PBS before incubation with Alexa Fluor 488 secondary antibody for 1 h at 37 °C. Finally, cells were incubated with DAPI for 15 min at 37 °C.
4
Apoptosis in Hepatic Stellate Cells Visualized
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Apoptotic HSCs were localized with labeled nucleotides in TUNEL staining. The mouse liver sections were stained according to the in situ cell death detection kit (Roche, Germany) protocol. The sections were then incubated with an α-SMA primary antibody (1:500, Abcam, United States) and a CY3 goat anti-rabbit fluorescence secondary antibody (1:300, Servicebio, Wuhan, China). The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and photographed under a fluorescence microscope. The relative number of apoptotic cells in each group was analyzed using Image-Pro Plus 6.0 software.
5
Quantifying Fibroblast α-SMA Expression
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Human conjunctival fibroblasts were lysed using 2x SDS sample buffer (100 mM Tris HCL ph6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, 0.2% bromophenol blue). Equal amounts of protein were loaded onto and run on 4–12% NuPAGE Bis-Tris protein gels (Novex, Life Technologies). The gels were transferred onto nitrocellulose blotting membranes (Amersham, Life Sciences), and blocked in 3% non-fat milk in PBST (PBS 0.1% Tween) for 1 hour. The membranes were then incubated overnight at 4 °C in αSMA primary antibody (1:200, polyclonal rabbit, ab5694, Abcam). The next day, the membranes were washed three times for 10 minutes in PBST, and incubated for 1 hour at room temperature in anti-rabbit HRP (1:2500, Dako). The membranes were then washed three times for 10 minutes in PBST, treated with ECL solution (Amersham, Life sciences) for 5 minutes, and scanned on an Odyssey IR Imager (LI-COR).
6
Immunohistochemistry Staining for α-SMA
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For immunohistochemistry staining (IHC), the harvested sections were deparaffinized and rehydrated. Subsequently, the deparaffinized sections were processed with secondary antibody and ABC complex following incubation with the α-SMA primary antibody (1:250, Abcam). Finally, the samples were visualized by the chromogenic substrate diaminobenzidine (DAB) substrate. An optical microscope (Olympus IX 70, Tokyo, Japan) was applied to obtain images of the stained sections.
7
Immunofluorescent Staining of ARPE-19 Cells
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The ARPE-19 cells were seeded on glass coverslips at a density of 5 × 104 cells per well in 24-well culture plates. After the appropriate culture time, the cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 in PBS for 15 min. After blocking with normal goat serum working solution for 20 min, the cells were incubated with α-SMA primary antibody (1:100 dilution; Abcam, Cambridge, UK) and p65 (1:50 dilution; Cell Signaling Technology, Beverly, MA) overnight. After washing with PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody was added and incubated for 1 h in the dark. Cells were washed and then incubated for 10 min with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) to stain the nuclei. Images were captured using a fluorescence microscope (Nikon Eclipse Ti-E) at 200X magnification. Images were processed in ImageJ.
8
Immunofluorescent Quantification of α-SMA
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The hHSCs were grown in 12-well plates and treated with DMEM (5% FBA; 1% P/S) in the absence or presence of 5.0 mM VPA for 6 h and 4 days. The cells were washed with PBS buffer and incubated in 3% sheep serum at room temperature for 1 h, Next, α-SMA primary antibody (rabbit, diluted 1:100, ab5694; Abcam, Cambridge, UK) overnight at 4 °C, and then washed with PBS buffer. Secondary antibody (goat anti-rabbit, diluted 1:250, A11008; Life Technologies, Carlsbad, CA, USA, Alexa Fluor@488) was added to the wells and incubated 20 min at room temperature. Finally the pictures of the cells were obtained with a ZOETM Fluorescent Cell Inager (BIO RAD, 742BR1669, Singapore).
9
α-SMA Expression in PDGF-Stimulated HSCs
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HSCs were cultured overnight in DMEM supplemented with 10% FBS. PDGF (10 ng/mL) was then added with 10 mg/mL KXRG and the cells were seeded in two-well chamber slides and cultured for an additional 24 h. Thereafter, the cells were fixed in 4% formaldehyde containing 2% sucrose for 10 min and permeabilized with phosphate-buffered saline containing 0.5% Triton X-100 (Sigma-Aldrich) for 5 min. The α-SMA primary antibody (Abcam) was diluted at 1 : 200, and the secondary antibody (HRP-conjugated goat anti-rat IgG) (Abcam) was diluted at 1 : 1000. Positive cells were counted after color reaction of DAB using an optical microscope (Olympus, Tokyo, Japan).
10
Immunofluorescence analysis of P1-MEF-HFSC cells
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The culture medium from P1-MEF-HFSC cells was discarded, the cells were washed with PBS and fixed with 4% polyformaldehyde for 15 min at room temperature. Cells were washed with PBS and treated with 0.3% Triton after 20 min. The samples were washed in PBS and blocked with 10% goat serum (cat. no. ab7481; Abcam) for 1 h at room temperature, followed by an overnight incubation with 1:200 diluted K15, α-SMA primary antibody (cat. no. ab5694; Abcam) and NCAM monoclonal antibody (cat. no. ab204446; Abcam) at 4˚C. Cells were washed with PBS and treated with secondary antibodies [1:500; Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488); cat. no. ab150077; 1:500; Goat Anti-Rabbit IgG H&L (TRITC); cat. no. ab7087; Abcam] at 37˚C for 30 min. The nucleus was stained with DAPI for 10 sec at 37˚C. Images were acquired using a Leica immunofluorescences microscope (Leica Microsystems, Inc.; magnification, x40).
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