Cells or exosomes were collected and denatured. Proteins were separated by HEPES‐Tris gel and transferred onto polyvinylidene difluoride membranes. After blocking with 5% skimmed milk for 90 min, membranes were probed with CD9 antibody (1:500) (Proteintech, MA, USA), CD63 antibody (1:200) (Proteintech, MA, USA), TSG101 antibody (1:1000) (Proteintech, MA, USA), calnexin antibody (1:1000) (ABclonal, China), αSMA antibody (1:5000) (Proteintech, MA, USA), NOX4 antibody (1:1000) (Proteintech, MA, USA), Smad2 + Smad3 antibody (1:1000) (Abcam, USA), Phospho‐Smad2‐S465/467 + Smad3‐S423/425 antibody (1:1000) (ABclonal, China) and β‐actin antibody (1:5000) (ZSGB‐Bio, China) at 4°C overnight, followed by incubation with horseradish peroxidase‐linked secondary antibodies at room temperature for 1.5 h. Protein bands were visualized using the BeyoECL Plus kit (Beyotime, China). Finally, the membranes were exposed to a gel imaging system ChemiDox XRS + System. Gray values of protein blots were calculated using ImageJ software v.1.8.0 software. β‐actin served as the internal control.
αsma antibody
Manufactured by Proteintech
Sourced in United States, China
The αSMA antibody is a primary antibody that specifically recognizes the alpha-smooth muscle actin (αSMA) protein. αSMA is a cytoskeletal protein that is commonly used as a marker for the identification of myofibroblasts and activated smooth muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
Nox4 antibody, by Proteintech (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
β actin antibody, by ZSGB-BIO (1 mentions)
Cd9 antibody, by Proteintech (1 mentions)
Cd63 antibody, by Proteintech (1 mentions)
Tsg101 antibody, by Proteintech (1 mentions)
Gfp antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Stat3 antibody, by Proteintech (1 mentions)
Col1 antibody, by Proteintech (1 mentions)
Goat anti rabbit igg fitc, by Santa Cruz Biotechnology (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Hif 1α antibody, by Proteintech (1 mentions)
Rhodamine conjugated goat anti rat igg h l, by Proteintech (1 mentions)
Coralite 488 conjugated anti rabbit igg h l, by Proteintech (1 mentions)
β catenin antibody, by Proteintech (1 mentions)
Bx53 biomicroscope, by Olympus (1 mentions)
Bca protein concentration kit, by Beyotime (1 mentions)
8 protocols using αsma antibody
1
Western Blot Analysis of Exosomal Markers
2
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were fixed, embedded in paraffin, and sectioned at a thickness of 4 μm. After deparaffinization and rehydration, sections were covered with Tris‐EDTA (pH 9.0) buffer for antigen retrieval and blocked with 3% H2O2 for 20 min. Next, sections were blocked with 5% goat serum for 30 min and incubated with αSMA antibody (1:1500) (Proteintech, MA, USA) and p16INK4a (ZSGB‐Bio, China) at 4°C overnight, followed by incubation with horseradish peroxidase‐labeling secondary antibody at 37°C for 30 min. Blots were visualized with 3,3′‐diaminobenzidine (DAB) solution and counterstained with hematoxylin. Images were taken under an Olympus BX51 microscope with ×100 and ×200 objectives. Stained slides were reviewed and scored using ImageJ software.
3
Immunofluorescence Staining Protocol
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The cells were fixed with 4% paraformaldehyde, permeabilized in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, and blocked in 10% goat serum albumin in PBS. The cells were incubated with one of the following antibodies: Col1 antibody (1:500, Proteintech, China), α-SMA antibody (1:200, Proteintech, China), STAT3 antibody (1:500 in PBS, Proteintech, China), GFP antibody (1:100, Proteintech, China), in combination with goat anti-rabbit secondary antibody conjugated with CoraLite594 (1:500, Proteintech, China). Nuclei were counterstained with DAPI (Solarbio, China).
4
Pericyte Coverage and Tumor Hypoxia Evaluation
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Tissues were fixed in 4% paraformaldehyde for 24 hours and paraffin-embedded, sectioned, dewaxed in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed in citric acid buffer (pH 6.0). Sections were blocked in 2% normal goat serum for 1 hour and stained with primary antibodies as follows: anti-CD31 antibody (1:500; Abcam) for endothelium, α-SMA antibody (1:100; Proteintech) for pericytes, and HIF-1α antibody (1:50; Proteintech) for hypoxia. The sections were then washed and incubated with rhodamine-conjugated goat anti-rat IgG (H + L) (1:50; Proteintech) or goat anti-rabbit IgG-FITC (1:200; Santa Cruz) for 40 min at RT. The level of pericyte coverage was presented as a percent of the length along CD31+ vessels. The ratio of hypoxia in the tumour sections was presented as HIF-1α staining area/CD31+ area.
5
Evaluating miR-9-5p Effects on Myofibroblast Transformation
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HGF‐1 cells were seeded onto 12‐well plates containing 15‐mm diameter glass coverslips. HGF‐1 cells were transfected with 100 nM miR‐9‐5p mimic or mimic NC. Then cells were incubated with 5 ng/ml TGF‐β1 for 48 h. Then treated cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.25% Triton X‐100 in PBS for 20 min at room temperature. Cells were washed with PBS, blocked with 1% BSA in PBS for 1 h, and incubated overnight at 4°C with αSMA antibody (1:200) (Proteintech, MA, USA) and NOX4 antibody (1:100) (Proteintech, MA, USA). Nuclear staining was performed with DAPI (Beyotime, China). Then, Coralite 594‐conjugated anti‐mouse IgG (H+ L) (1:200) (Proteintech, MA, USA) and Coralite 488‐conjugated anti‐rabbit IgG (H+L) (1:200) (Proteintech, MA, USA) were used as secondary antibodies. The coverslips were mounted on slides using an anti‐fluorescence quenching sealing agent (Beyotime, China). An Olympus BX51 microscope was used to visualize cell fluorescence at the appropriate excitation wavelength for the fluorophore.
6
Immunofluorescence Staining for Tissue Analysis
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Immunofluorescence staining was previously described.30 Briefly, after rehydrated with gradient ethanol, sections were repaired with EDTA solution at 100°C for 15 minutes and then washed by PBS solution 3 times. Non‐specific binding sites were blocked by incubation with goat serum at room temperature for 30 minutes. After blocking, sections were incubated with the primary antibodies overnight at 4°C and with a secondary fluorescent antibody for 1 hour at 37°C, respectively. Then, sections were counter‐stained with DAPI (Beyotime Biotechnology) and evaluated by a fluorescent microscope (Olympus bx53 biomicroscope). The following primary antibodies were used: α‐SMA antibody (Proteintech), Collagen Type I (Collagen I) antibody (Proteintech) and β‐Catenin antibody (Proteintech).
7
Western Blot Analysis of EMT Markers
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Cells were collected and added to RIPA lysate buffer (plus 100:1 phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor) for protein extraction, and a bicinchoninic acid (BCA) protein concentration kit (Beyotime, Jiangsu, China) was used to determine the protein concentrations. Equal amounts of protein samples were subjected to SDS-PAGE, transferred to nitrocellulose (NC) filter membranes, and blocked using 5% skim milk powder. After washing the membranes, α-SMA antibody (Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; Affinity Biosciences, Cincinnati, OH, USA; AF0131), N-cadherin (1:5000; Abcam ab76011, Cambridge, MA, USA), FAK (1:1000; Abcam ab40794), P-FAK (1:1000; Abcam ab81298), and glyceraldehyde-3-phosphate (GAPDH) (1:5000; Shanghai Dianyin Biotechnology Co., Ltd., Shanghai, China) antibodies were incubated overnight at 4°C. The membranes were washed again and incubated with secondary antibody (EarthOx Life Sciences, Millbrae, CA, USA) for 1 hour at room temperature. The membranes were washed and detected using an ODYSSEY fluorescence imaging system (LI-COR, Lincoln, NE, USA). Finally, the OD values for each group were analyzed using ImageJ image analysis software (National Institutes of Health, Bethesda, MD, USA).
8
Quantifying Lung Tissue Remodeling in Mice
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The lung tissue samples of mice were fixed in 10% formalin, embedded in paraffin and then sliced into 3‐4 μm sections. The lung sections were stained with hematoxylin and eosin. They were subjected to immunostaining procedures and Masson staining as previously described.14 The area of peribronchial Masson trichrome staining (blue colour) was visualized and quantified. The α‐SMA protein was identified in the paraffin‐embedded sections of the lung tissue by immunohistochemical staining with an α‐SMA antibody (Proteintech). The sections were examined under a light microscope and the data were quantified using Image Pro 6.0 software. Results are expressed as the area of α‐SMA staining per micrometer length of bronchioles’ basement membrane of 150‐200 μm in internal diameter.
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