Immunohistochemical staining was performed as previously described, with a slight modification [29 (link)]. A GTVisionTM III Detection System/Mo & Rb (Gene Tech, Shanghai, China) was used following the manufacturer’s instructions. All antibodies were freshly diluted with 2% goat serum (Boster, Wuhan, China). The LYRIC (Mtdh) antibody (Abcam, Cambridge, Britain) was used at a 1:100 dilution, the fibronectin antibody (BD Biosciences, USA) was used at a 1:200 dilution, the α-SMA antibody (Sigma-Aldrich, Missouri, USA) was used at a 1:250 dilution and the E-cadherin antibody (BD Biosciences, California, USA) was used at a 1:100 dilution. Slides were counterstained with haematoxylin followed by a bluing reagent. Negative controls were treated with PBS instead of primary antibodies.
Fibronectin antibody
Manufactured by BD
Sourced in United States
Fibronectin antibody is a laboratory reagent used to detect and quantify the presence of fibronectin, a glycoprotein found in the extracellular matrix of various tissues. It can be used in techniques such as Western blotting, immunohistochemistry, or ELISA to study the expression and distribution of fibronectin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
α sma antibody, by Merck Group (2 mentions)
Gtvisiontmiii detection system mo rb, by Gene Tech (1 mentions)
Goat serum, by Boster Bio (1 mentions)
E cadherin antibody, by BD (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Ve cadherin, by BD (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Total and phospho cofilin, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by Welgene (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Recombinant human bmp 7, by R&D Systems (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Tgf β, by R&D Systems (1 mentions)
T smad3, by Cell Signaling Technology (1 mentions)
Il 6 antibody, by Novus Biologicals (1 mentions)
Recombinant tnf α, by R&D Systems (1 mentions)
T smad2, by Cell Signaling Technology (1 mentions)
T nf κb, by Cell Signaling Technology (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
4 protocols using fibronectin antibody
1
Immunohistochemical Staining of LYRIC, Fibronectin, α-SMA, and E-cadherin
2
Molecular Mechanisms of EGCG in Endothelial Cells
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EGCG (≥95% by HPLC) from green tea was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in water. The antibodies used for Western blotting were as follows: fibronectin antibody was purchased from BD Pharmingen; CD31, VE-cadherin, β-actin, total- and phospho-cofilin, total- and phospho-cell division cycle (CDC) 42, RhoA, and total- and phospho-smad2/3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); total- and phospho-NF-κB p65 and total- and phospho-inhibitor of NF (I)κBα antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and α-smooth muscle actin (SMA) antibody was purchased from Abcam (Cambridge, UK).
3
Antibody Characterization in Cell Signaling
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The source for antibodies used in this study was as follows. Dulbecco's modified Eagle medium (DMEM):F12 and penicillin-streptomycin were obtained from Welgene, Inc. (Daegu, Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) was obtained from Invitrogen (Carlsbad, CA, USA). Recombinant human BMP7, recombinant TNF-α, IL-1β, and TGF-β were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies for phosphorylated (p)-SMAD1/5/8, total (t)-SMAD1/5/8, p-SMAD2, t-SMAD2, p-SMAD3, t-SMAD3, p-extracellular signal-related kinase (ERK), t-ERK, p-p38, t-p38, TNF receptor–associated factor 6 (TRAF6), intercellular adhesion molecule 1 (ICAM-1), p-nuclear factor (NF)-κB, and t-NFκB, p-Akt, t-Akt, p-Jun N-terminal kinase (Jnk), and t-Jnk were obtained from Cell Signaling Technology (Danvers, MA, USA). Fibronectin antibody was obtained from BD Bioscience (Franklin Lakes, NJ, USA), α-SMA antibody were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA), and IL-6 antibody was obtained from Novus Biologicals (Centennial, CO, USA). Collagen 1α and IL-8 antibodies were obtained from Abcam (Cambridge, MA, USA). BMP7 and β-actin antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies used in the study are listed in detail in Supplementary Table S1 .
4
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in a RIPA buffer (Boston Bio-Products, MA) in the presence of protease and phosphatase inhibitors (Thermo-Fisher Scientific, NY). Protein concentration was determined using the Bradford protein assay. Here, 20–30 ug of protein was loaded and separated by SDS-PAGE; then, it was transferred onto PVDF membranes (EMD Millipore, MA). Membranes were blocked in 5% milk in TBS-T for 1 h at room temperature and incubated overnight at 4∘C with primary antibodies. The membranes were washed and incubated with HRP-congregated antimouse or antirabbit secondary antibody (Bio-Rad, CA) for 1 h at room temperature. Proteins were detected using the ECL Western blotting substrate (Thermo-Fisher Scientific, NY). CDK4, CDK6, TAZ, YAP, E-cadherin, and vimentin antibodies were purchased from Cell Signaling Technology; Fibronectin antibody from BD Biosciences; Flag (M2) antibodies from Sigma-Aldrich; and anti-GAPDH from Ubiquitin-Proteasome Biotechnologies.
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