Anti fibronectin

Manufactured by Merck Group

Sourced in United States, China, Germany, Canada

Anti-fibronectin is a laboratory-grade reagent used in research and diagnostic applications. It is a monoclonal antibody that specifically binds to and detects the presence of fibronectin, a key extracellular matrix protein. This product can be utilized in various immunoassay techniques to measure and analyze fibronectin levels in biological samples.

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Lab products found in correlation

Anti α sma, by Merck Group (12 mentions) Anti laminin, by Merck Group (8 mentions) Anti β actin, by Merck Group (7 mentions) Anti e cadherin, by BD (7 mentions) Anti β catenin, by BD (7 mentions) Anti vimentin, by Merck Group (6 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions) F3648, by Merck Group (6 mentions) Anti active β catenin, by Cell Signaling Technology (5 mentions) Anti vimentin, by Abcam (5 mentions) Ab128568, by Abcam (4 mentions) Anti α tubulin, by Merck Group (4 mentions) Apotome, by Zeiss (4 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (4 mentions) Anti flag, by Merck Group (3 mentions) Ab186735, by Abcam (3 mentions) Ab26350, by Abcam (3 mentions) Triton x 100, by Merck Group (3 mentions) Anti collagen 1, by Boster Bio (3 mentions) Anti collagen 1, by Southern Biotech (3 mentions)

91 protocols using anti fibronectin

Immunophenotyping of Aortic Cell Populations

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Aortic sections were immunostained with anti-laminin γ1 (Abcam), anti-nestin (Sigma), anti-SMA (Sigma), anti-SM22α (GeneTex), anti-fibronectin (Millipore), anti-collagen I (Sigma), anti-collagen IV (Millipore), anti-CD90 (Abcam), anti-CD105 (Biolegend), anti-PDGFRα (eBiosciences), anti-c-Kit (R&D), and anti-Ki67 (Millipore) antibodies overnight at 4°C. For fluorescent staining, sections were incubated with appropriate fluorescent secondary antibodies (Invitrogen) for 1 hour at room temperature. Due to strong autofluorescence in the green channel of aortic tissue, Alexa-647 (artificially colored in green) rather than Alexa-488 was used. For DAB staining, sections were incubated with biotinylated secondary antibodies for 1 hour at room temperature, followed by ABC kit (Vector) and DAB Kit (Vector), according to the manufacturer’s instructions. After mounting, the sections were examined and photographed with Zeiss Axiovert 200 or Leica confocal microscope.

Yao Y., Norris E.H, & Strickland S. (2014). The cellular origin of laminin determines its role in blood pressure regulation. Cellular and molecular life sciences : CMLS, 72(5), 999-1008.

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Immunohistochemical Analysis of Gastric Tissue

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Stomachs from 30- to 90-day-old Bmpr1a^ΔMES and control littermates were fixed, sectioned and stained as previously described6 (link)17 (link) or according to the manufacturer’s protocol (395B-1KT Sigma-Aldrich). Immunostainings were performed as previously described6 (link)59 (link). The following antibodies were used at the indicated dilutions: anti-PCNA (1:10,000, Abcam), anti-Bmpr1a (1:300, Abcam), anti-pSmad1-5-8 Ser463-465 (1:500, Santa Cruz), 488 Alexa Fluor Lectin GSII (1:200, Invitrogen) anti-H+/K+-ATPase (1:2, MBL), anti-GIF (1:75, Abnova), anti-somatostatin (1:100, Santa Cruz), anti-gastrin (1:100, Chemicon), anti-CD3 (1:200, Dako), anti-F4/80 (1:100, eBioscience), anti- α-Sma clone 1A4 (1:5000, Sigma), anti-Vimentin^XP D21H3 (1:100, Cell Signaling), anti-fibronectin (1:1000, Millipore), anti-collagen-I (1:200, Thermo Scientific), anti-collagen-IV (1:200, Chemicon International) and FITC-conjugated anti-rabbit IgG (1:300, Vector), FITC-conjugated anti-goat IgG (1:300, Vector) and Alexa 568-conjugated anti-mouse (1:400, Invitrogen). For immunofluorescence, images were captured on a Leica DMLB2 microscope using a Leica DC300 camera except for α-Sma/pSmad1-5-8 immunostaining and α-Sma/Bmpr1a where a Confocal Zeiss LSM700 along with Zen Blue software was used. For IHC, images were captured on a Nanozoomer Digital slides Scanner (Hamamatsu).

Roy S.A., Allaire J.M., Ouellet C., Maloum-Rami F., Pomerleau V., Lemieux É., Babeu J.P., Rousseau J., Paquet M., Garde-Granger P., Boudreau F, & Perreault N. (2016). Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade. Scientific Reports, 6, 32759.

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Protein Separation and Western Blot Analysis

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Total protein, cytoplasmic protein and mitochondrial protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore) followed by blocking and immunoblotting with various primary antibodies, including anti-RCAN1, anti-FLAG (Sigma), anti-COX4, anti-Drp1, anti-Fis1, anti-Mfn2, anti-Opa1 (all Cell Signaling Technology, Boston, U.S.A.), anti-fibronectin (Millipore), anti-collagen I, anti-β-tubulin, anti-β-actin (Santa Cruz Biotechnology, Dallas, U.S.A.). After overnight incubation at 4°C, the membranes were immersed in a solution including appropriate secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots were developed using ECL kit (Millipore).

Chen H.M., Dai J.J., Zhu R., Sang X.Y., Peng F.F., Yu H, & Zhang B.F. (2020). RCAN1.4 mediates high glucose-induced matrix production by stimulating mitochondrial fission in mesangial cells. Bioscience Reports, 40(1), BSR20192759.

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Western Blot Analysis of Cell Signaling

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Cell pellets were lysed by adding 2× SDS sample buffer. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose and stained with 0.05% Ponceau S (Sigma) to ensure equivalent protein loading. Immunoblots were blocked with 5% bovine serum albumin and probed overnight at 4°C with anti-CLIC1 (Abcam), anti-fibronectin (Millipore), anti-Slug (Cell Signaling), anti-MLCK (Sigma), anti-phospho-MLCK (Life Technologies), anti-β3 (BD Biosciences), and anti-β-Actin (Sigma) antibodies followed by incubation with peroxidase-conjugated anti-mouse or rabbit IgG antibody (Biorad). Lastly, the blots were incubated with enhanced chemiluminescence (Perkin Elmer) to visualize antibody binding.

Gurski L.A., Knowles L.M., Basse P.H., Maranchie J.K., Watkins S.C, & Pilch J. (2014). Relocation of CLIC1 Promotes Tumor Cell Invasion and Colonization of Fibrin. Molecular cancer research : MCR, 13(2), 273-280.

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Fibrin-Embedded Cell Imaging and Analysis

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Fibrin-embedded cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% triton ×100 and incubated with anti-fibronectin (Millipore), anti-CLIC1 (Abcam), anti-β3 integrin (Abcam), anti-pMLC (Cell Signaling) or isotype control, followed by incubation with Alexa Fluor 488- or 546-conjugated secondary antibody (Invitrogen). To visualize the cytoskeleton and nuclei, cells were stained with Alexa Fluor 546-phalloidin (Invitrogen) and Draq5 (eBioscience), respectively. The myosin light chain kinase (MLCK) inhibitor ML-7 was added at 10 µM where indicated. Fibrin-embedded cells were analyzed using a confocal microscope (Leica TCSSL) and images were processed with Adobe Photoshop. To quantify fibronectin matrix formation, fibrin-embedded cells were stained for fibronectin as described except that the cells were not permeabilized. 20× confocal images were analyzed for the % area of fibronectin staining using ImageJ software. ImageJ was also used to assess CLIC1 expression in invadopodia and the cell periphery, which were identified using F-actin staining. To this end, we measured the average CLIC1 fluorescence intensities in the cell periphery/invadopodia compared to the cell interior (without the nucleus) and calculated the ratio between the two compartments.

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Osteogenic Differentiation of Mesenchymal Cells

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Bovine fibrinogen, thrombin, aminocaproic acid, L-Ascorbic acid and glycerol 2-phosphate were purchased from Sigma (St. Louis, MO). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA). Fetal bovine serum (FBS), HEPES buffer solution, and penicillin-streptomycin solution were from GibcoBRL (Carlsbad, CA), and ascorbic acid-free α-MEM was from WelGene (Daegu, Korea). The MicroBCA assay kit was from Pierce-Thermo (Rockford, IL). Qunat-iT PicoGreen dsDNA-assay kit was from Invitrogen (Eugene, OR). West-Zol was from Intron Biotechnology (Seoul, Korea). A Dual-GloTM Luciferase Assay System was from Promega (Madison, WI). Protease inhibitor cocktail tablets (Complete) were from Roche (Basel, Switzerland). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-fibronectin and anti-vitronectin antibodies were from Chemicon (Temecula, CA).

Oh J.H., Kim H.J., Kim T.I, & Woo K.M. (2014). Comparative evaluation of the biological properties of fibrin for bone regeneration. BMB Reports, 47(2), 110-114.

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Immunofluorescence Imaging of U87 Cells

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U87 control and shTMEM230 cells were fixed with 4% Paraformaldehyde (Sigma) in 1× PBS for 10 min at room temperature (RT). Cells were incubated with a blocking buffer of 5% normal goat serum in 1× PBS. The primary antibodies used were anti-FIBRONECTIN (1:200 dilution, Chemicon International, AB2033), anti-CAVEOLIN-1 (1:100 dilution, Santa Cruz, sc-7875) and anti-phalloidin-TRITC conjugated (1:2,000, Sigma, P1951), all incubated for 2 hours at RT. Both were incubated for 2 h at RT. The cells were washed with 1× PBS and incubated with a secondary antibody of goat anti-rabbit Alexafluor-555 (1:500 dilution, A21429, Life Technologies) for 1 h at RT. Nuclei were visualized with 4′,6′-diamidino-2-phenylindole (DAPI) staining.

Cocola C., Magnaghi V., Abeni E., Pelucchi P., Martino V., Vilardo L., Piscitelli E., Consiglio A., Grillo G., Mosca E., Gualtierotti R., Mazzaccaro D., La Sala G., Di Pietro C., Palizban M., Liuni S., DePedro G., Morara S., Nano G., Kehler J., Greve B., Noghero A., Marazziti D., Bussolino F., Bellipanni G., D’Agnano I., Götte M., Zucchi I, & Reinbold R. (2021). Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy. Frontiers in Cellular Neuroscience, 15, 703431.

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Western Blot Analysis of Protein Markers

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Protein expression was analyzed by Western blot analysis as described previously.^{12 (link)} The primary antibodies used were as follows: anti-fibronectin (F3648, Sigma-Aldrich), anti-desmin (PB0095; Boster, Wuhan, China), anti-vimentin (SAB1305447, Sigma-Aldrich), anti-MMP-9 (AB805; Millipore), anti-podocin (sc-22298; Santa Cruz Biotechnology, Santa Cruz, CA), anti-podocalyxin (AF1556; R&D Systems), anti-ZO-1(QF215185; Life technologies), anti-nephrin(ab58968; Abcam), anti-Klotho (AF1819; R&D Systems), anti-p47^phox (07-500; Millipore, Billerica, MA), anti-Nox2 (BA2811; Boster), anti-active β-catenin (05-665; Millipore), anti-Snail1 (ab17732; Abcam), anti-Wnt1 (ab15251; Abcam), anti-Wnt7a (sc-26361; Santa Cruz Biotechnology), anti-Flag(F3165; Sigma-Aldrich), anti-p65(4764S; Cell Signaling Technology), anti-p-p65(3031S; Cell Signaling Technology), anti-α-Tubulin (RM2007; Ray Antibody, Beijing, China) and anti-β-actin (MAB1501; Millipore, Billerica, MA).

Zhou L., Chen X., Lu M., Wu Q., Yuan Q., Hu C., Miao J., Zhang Y., Li H., Hou F.F., Nie J, & Liu Y. (2019). Wnt/β-catenin links oxidative stress to podocyte injury and proteinuria. Kidney international, 95(4), 830-845.

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Immunofluorescence Staining of HNSCC

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Human head and neck squamous cell carcinoma samples were obtained from the Royal Marsden Hospital after informed consent through the project CCR2924 (Approved by the London/Chelsea NHS REC with code REC: 06/Q0403/125). All material was handled according to relevant ethical regulations by the Human Tissue Act. After excision the tumours were flash-frozen. Sections were stained as previously described ^{46 (link)}. Briefly, fresh frozen sections were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and stained with the following antibodies: anti-fibronectin (dilution 1/500, Sigma, F3648), anti-vimentin (dilution 1/100, Sigma,V2258), anti-TFAP2C (dilution 1:100, Abacm#218107), anti-αSMA (dilution 1/200, Cy3-conjugated clone#1a4, Sigma, C6198) and 633-phalloidin (dilution 1:500, Sigma, 68825). Sections were mounted using MOWIOL reagent and imaged using a Zeiss LSM 780 microscope.

Park D., Wershof E., Boeing S., Labernadie A., Jenkins R.P., George S., Trepat X., Bates P.A, & Sahai E. (2019). Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions. Nature materials, 19(2), 227-238.

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Immunofluorescence Analysis of Muscle Proteins

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For TA, DIAPH, and EDL immunofluorescence, cryosections of 10 µm were fixed in 4% paraformaldehyde (Winkler), permeabilized with PBS-0.05% Triton, and blocked for 1 h with blocking buffer (BSA 2%, 0.05% Triton X-100 in PBS). Samples were incubated overnight at 4 °C with: anti-Fibronectin (F3648, Sigma Aldrich), anti-Collagen I (PA1-26204, Invitrogen, Waltham, MA, USA), anti-Slow Myosin (M8421, Sigma Aldrich), anti- nNOSµ (617000, Invitrogen) and anti-IIA myosin heavy chain antibody (SC-71, DSHB, University of Iowa, Iowa City, IA, USA). The corresponding Alexa Fluor- 568 or 488-conjugated anti-IgGs (Invitrogen) were used as secondary antibodies. In addition, fluorescent wheat germ agglutinin (WGA) (Thermo Fisher, Waltham, MA, USA) was used to label cell membranes and Hoescht for nuclear staining. Slices were washed and mounted in Fluoromount G. Samples were visualized on a Nikon Eclipse E600 using NIS Elements software v4.20 or Leica DM2000 epifluorescence using Mshot Image Analysis System software with 20× or 40× objectives.

Cáceres-Ayala C., Mira R.G., Acuña M.J., Brandan E., Cerpa W, & Rebolledo D.L. (2023). Episodic Binge-like Ethanol Reduces Skeletal Muscle Strength Associated with Atrophy, Fibrosis, and Inflammation in Young Rats. International Journal of Molecular Sciences, 24(2), 1655.

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