Cells were cultured on coverslips and were fixed with 4% paraformaldehyde for 20–30 min, permeabilized by 0.4% Triton X‐100, and blocked in 1% BSA/1x PBST at room temperature for 1 h. Primary and secondary antibodies were diluted in a ratio of 1:1000 and 1:10,000 with blocking buffer, respectively. The information of primary antibodies used in current study was listed as following: vimentin (ab24525, Abcam), α‐SMA (A2547, Sigma), collagen I (C2456, Sigma), FN1 (ED‐A) (ab6328, Abcam), MMP1 (ab137332, Abcam), MMP2 (GTX104577, GeneTex), MMP8 (GTX61732, GeneTex), MMP9 (ab38898, Abcam), and MMP13 (ab39012, Abcam). For secondary antibodies: anti‐rabbit Dylight 488 (SA5‐10038, Thermo Fisher Scientific, Waltham, MA), anti‐chicken Dylight 550 (SA5‐10071, Thermo Fisher Scientific), anti‐mouse Dylight 633 (35512, Thermo Fisher Scientific), and mouse Dylight 488 (35503, Thermo Fisher Scientific). Samples were immunostained with primary antibody (1:200) at 4°C overnight, and further incubated with secondary antibodies (1:200) conjugated with Dylight at room temperature for 1 h. Nuclei were counterstained with DAPI (1:1000), and the samples were mounted with anti‐fade solution. Fluorescent images were obtained using Leica TCS SP8 confocal microscope and analyzed by ImageJ software to determine the fluorescent intensity.78 , 79 , 80
Gtx104577
Manufactured by GeneTex
Sourced in Cameroon
GTX104577 is a laboratory equipment product. It is a device used for the specific purpose of [PRODUCT FUNCTION]. The core function of this product is to [PRODUCT CORE FUNCTION]. No further details or interpretations are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab39012, by Abcam (1 mentions)
Ab24525, by Abcam (1 mentions)
Ab137332, by Abcam (1 mentions)
Ab6328, by Abcam (1 mentions)
Anti rabbit dylight 488, by Thermo Fisher Scientific (1 mentions)
Anti mouse dylight 633, by Thermo Fisher Scientific (1 mentions)
C2456, by Merck Group (1 mentions)
A2547, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Ab38898, by Abcam (1 mentions)
Ab181340, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab181606, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Proteinase inhibitor, by Beyotime (1 mentions)
Ab207178, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Gtx100619, by GeneTex (1 mentions)
4 protocols using gtx104577
1
Immunofluorescence Staining of Cellular Markers
2
Western Blot Analysis of Exosomal Proteins
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Protein samples of exosomes and cells were lysed in RIPA buffer (Beyotime) supplemented with proteinase inhibitors (Beyotime). The protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime). Equal amounts of proteins (10 µg) were separated on an 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel and transferred onto polyvinylidene fluoride (PVDF; Millipore) membranes. The membranes were then blocked in 5% (w/v) non-fat milk and incubated with the primary antibodies overnight at 4°C. The following antibodies were used: CD9 (1:1,000, ab92726; Abcam), CD63 (1:1,000, ab134045; Abcam), heat shock protein (HSP)70 (1:1,000, ab181606; Abcam), Piwil2 (1:1,000, ab181340; Abcam), matrix metalloproteinase (MMP)9 (1:1,000 ab76003; Abcam), MMP2 (1:1,000, GTX104577; Genetex), α-smooth muscle actin (α-SMA; 1:1,000, ab32575; Abcam), Vimentin (1:1,000, GTX100619; Genetex), fibroblast-activating protein (FAP; 1:1,000, ab207178; Abcam), GAPDH (1:1,000, ab181602; Abcam). Finally, the membranes were incubated with HRP-conjugated secondary antibody (1:1,000, ZB-2301, ZB-2305; Zhongshan) for 2 h at room temperature. The immunoblots were visualized using the Immobilon Western Chemiluminescent HRP Substrate, and the bands were quantified, relative to GAPDH, by densitometric analysis (GeneGnome, Syngene UK).
3
Immunohistochemical Analysis of RAGE, TGF-β1, MMP2, and MMP9
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4 μm thickness paraffin sections were pasted onto slides for conventional dewaxing treatment. The sections were microwaved in a citrate buffer for 15 min (pH 6.0), and endogenous peroxidase was blocked by hydrogen peroxide and goat serum. The slices were then incubated with polyclonal RAGE (1 : 200, Sc365154, Santa Cruz Biotechnology, United Kingdom), TGF-β1 (1 : 100, BS6152, Bioword Technology, Inc., China), MMP2 (1 : 200, GTX104577, GeneTex, North, America), and MMP9 (1 : 50, BM4089, Boster Biological Technology, China) antibodies overnight at 4°C. The equivalent concentrations of polyclonal nonimmune IgG were used as controls. Then, sections were incubated with secondary HRP-En Vision IgG antibody at room temperature for 20 min and followed by incubation with a streptavidin solution. Color development was carried out using 3,3-diaminobenzidine tetrahydrochloride.
4
Bronchial Epithelial Cell Culture Protocol
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Normal human bronchial epithelial cells (NHBE, Lonza) were maintained in Keratinocyte SFM basal medium supplemented with 5 μg/L human recombinant epithelial growth factor (EGF), 50 mg/L bovine pituitary extract (BPE), 5 mg/L insulin, and 25 nM hydrocortisone at 37°C in a 5% CO2 atmosphere. Cells were passaged every 3 days and plated for experimental treatment within 6 passages.
NaAsO2, montelukast, fluticasone, N-acetylcysteine, and BAY117082 were purchased from Sigma–Aldrich (United States). Antibodies against fibronectin (Sigma, F3648), MMP-2 (GeneTex, GTX 104577), GAPDH (GeneTex, GTX100118), SMAD2/3 (GeneTex, GTX111123), β-catenin (R&D, AF1329), N-Cadherin (GeneTex, GTX127345), and E-Cadherin (GeneTex, GTX100443) were used.
NaAsO2, montelukast, fluticasone, N-acetylcysteine, and BAY117082 were purchased from Sigma–Aldrich (United States). Antibodies against fibronectin (Sigma, F3648), MMP-2 (GeneTex, GTX 104577), GAPDH (GeneTex, GTX100118), SMAD2/3 (GeneTex, GTX111123), β-catenin (R&D, AF1329), N-Cadherin (GeneTex, GTX127345), and E-Cadherin (GeneTex, GTX100443) were used.
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