Cells seeded at density of 2.5 × 103 per cm2 in Lab-Tek II 8-well chamber slides (Nalge Nunc, Rochester, NY, USA) were fixed in 4% formaldehyde for 10 min at room temperature. After washing cells in PBS three times, cells were subsequently incubated with ice-cold methanol for 20 min at −20 °C. The following primary antibodies diluted 1:200 including mouse monoclonal anti-α-SMA and mouse monoclonal anti-MHC (Santa Cruz Biotechnology, Aarhus, Denmark) were applied. After reaction with primary antibodies at 4 °C overnight, the cells were washed with PBS three times. Alexa 488-conjugated goat anti-mouse secondary antibody (Life Technologies Europe, Naerum, Denmark) was used to detect the localization of different antibodies respectively, and cell nuclei were counterstained with Hoechst 33342. The images were acquired and processed using the microscope Axio Observer Z1 (Carl Zeiss, Göttingen, Germany).
Mouse monoclonal anti α sma
Manufactured by Santa Cruz Biotechnology
Sourced in Denmark
Mouse monoclonal anti-α-SMA is an antibody that specifically recognizes the alpha-smooth muscle actin (α-SMA) protein. α-SMA is a cytoskeletal protein found in smooth muscle cells and other cell types. This antibody can be used to detect and localize α-SMA in various applications such as immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Mouse monoclonal anti mhc, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Novus Biologicals (1 mentions)
Mouse monoclonal anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Eclipse e800 fluorescence microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Oregon green 488, by Thermo Fisher Scientific (1 mentions)
Nis elements d v 4, by Nikon (1 mentions)
Hoechst dye, by Merck Group (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
5 protocols using mouse monoclonal anti α sma
1
Immunofluorescence Staining for α-SMA and MHC
2
Adrenomedullin Receptor Expression in Lung Fibrosis
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To explore the potential clinical relevance of our findings in a rat model of lung fibrosis and the potential utility of PulmoBind in the imaging of PVD in human lung fibrosis, we determined adrenomedullin receptor expression in human lungs with and without idiopathic pulmonary fibrosis. Protein lysates of lung tissues were prepared and subjected to SDS-electrophoresis. Antibodies used include the mouse monoclonal anti-vimentin (1:500); the mouse monoclonal anti-RAMP2 (1:400); the mouse monoclonal anti-SM22α (1:50.000), and the mouse monoclonal anti-αSMA (1:50.000) all from Santa Cruz Biotechnology, Santa Cruz, CA. The rabbit polyclonal anti-CD31 (1:1000; Novus Biologicals, Littleton, CO) and a mouse monoclonal anti-GAPDH (1:50,000; Ambion, Austin TX). Detection was done using clarity western ECL substrate (Perkin Elmer, Waltham, MA). Images were acquired, and quantification analyses were performed using a ChemiDocMP system (BioRad, ON, Canada) and densitometric analyses of Western blot were performed using ImageJ software. Protein signal was normalized to GAPDH.
3
Immunocytochemical Analysis of EMT Markers
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For E-cadherin, α-SMA, tenascin-C, and CPI-17 immunocytochemistry, IEC-6 cells were
cultured in DMEM containing 10% FBS on 25-mm cover glass until they reached 50–55%
confluence. When the cultured cells reached 50–55% confluence, medium was replaced with
DMEM supplemented with 0.5% FBS containing TGF-β1 (10
ng/ml−1) for 7 days to induce EMT cells.
The cell morphology was observed every day with an inverted microscope. The medium was
changed every 2 days. The cells were washed three times with HBSS and fixed with 10%
neutral formalin. After preservation, the cells were washed three times with PBS,
permeabilized with Tween 20 (Calbiochem, Darmstadt, Germany), and incubated with blocking
buffer containing 5% normal goat serum for 1 hr. Cells were then washed and incubated with
purified mouse anti-E-cadherin (1:250) (BD Biosciences, Catalog no. 61081), mouse
monoclonal anti-α-SMA (1:250) (Santa Cruz Biotechnology, Catalog no. sc-32251), rabbit
polyclonal α-h tenascin-C (1:250), or goat polyclonal anti-CPI-17 (1:250) (Santa Cruz
Biotechnology) antibody overnight at 4°C. The specimens were washed and incubated with the
appropriate secondary antibodies (1:1,000) for 2 hr at room temperature in a dark chamber.
Nuclei were stained with DAPI (Molecular Probes). Images were obtained using an Eclipse
E800 fluorescence microscope (Nikon, Tokyo, Japan).
cultured in DMEM containing 10% FBS on 25-mm cover glass until they reached 50–55%
confluence. When the cultured cells reached 50–55% confluence, medium was replaced with
DMEM supplemented with 0.5% FBS containing TGF-β1 (10
ng/ml−1) for 7 days to induce EMT cells.
The cell morphology was observed every day with an inverted microscope. The medium was
changed every 2 days. The cells were washed three times with HBSS and fixed with 10%
neutral formalin. After preservation, the cells were washed three times with PBS,
permeabilized with Tween 20 (Calbiochem, Darmstadt, Germany), and incubated with blocking
buffer containing 5% normal goat serum for 1 hr. Cells were then washed and incubated with
purified mouse anti-E-cadherin (1:250) (BD Biosciences, Catalog no. 61081), mouse
monoclonal anti-α-SMA (1:250) (Santa Cruz Biotechnology, Catalog no. sc-32251), rabbit
polyclonal α-h tenascin-C (1:250), or goat polyclonal anti-CPI-17 (1:250) (Santa Cruz
Biotechnology) antibody overnight at 4°C. The specimens were washed and incubated with the
appropriate secondary antibodies (1:1,000) for 2 hr at room temperature in a dark chamber.
Nuclei were stained with DAPI (Molecular Probes). Images were obtained using an Eclipse
E800 fluorescence microscope (Nikon, Tokyo, Japan).
4
Immunofluorescence Staining for α-SMA and Fibronectin
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The cells were grown on coverslips and treated as described above. Immunofluorescence staining was performed as described previously96 (link),97 (link). Briefly, the cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS at 25 °C for 15 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS at 25 °C for 15 min and then washed with PBS. Thereafter, non-specific bindings were blocked with 1% BSA in PBS at 25 °C for 30 min. The cells were incubated with mouse monoclonal anti-α-SMA (Santa Cruz Biotechnology; Santa Cruz, CA) or mouse monoclonal anti-fibronectin antibody (Santa Cruz Biotechnology) (diluted 1:50 in 1% BSA/PBS) or phalloidin conjugated with Oregon Green 488 (Invitrogen; Eugene, OR) at 37 °C for 1 h. The cells were washed three times with PBS and further incubated with Alexa Flour 488-conjugated donkey anti-mouse IgG antibody (diluted 1:500 in 1% BSA/PBS) (Invitrogen) mixed with Hoechst dye (Sigma-Aldrich) (for nuclear staining) (diluted 1:1000 in 1% BSA/PBS) at 37 °C for 1 h. Finally, the coverslips were mounted onto glass slides using 50% glycerol in PBS and the fluorescence images were captured under a fluorescence microscope (Eclipse 80i) (Nikon). Quantitative data were obtained and analyzed from at least 100 cells in ≥10 random HPFs for each sample using NIS-Elements D V.4.11 (Nikon).
5
Exosome Protein Profiling by Western Blot
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Exosomes and cells were lysed with RIPA buffer (Millipore) supplemented with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). The protein concentrations were determined using the BCA assay kit. The appropriate amount of protein lysates was loaded and separated by SDS-PAGE, transferred to 0.2-μm PVDF membranes, and incubated with primary antibodies of CD9, CD81, CD63, FAP (Abcam), mouse monoclonal anti-α-SMA (Santa Cruz), FSP, TSP-1 (DAKO), and Tn-C (1: 200 dilution; Abcam, Cambridge, MA), respectively. After incubation with the secondary anti-mouse and anti-rabbit antibodies (Cell Signaling), the protein bands were visualized using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK).
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