Ab6584

Manufactured by Abcam

Sourced in United States

Ab6584 is a laboratory equipment product. It is a type of device used for scientific research and analysis in a laboratory setting. The core function of this product is to facilitate the measurement and analysis of samples or materials.

Automatically generated - may contain errors

Lab products found in correlation

Nb600 408, by Abcam (2 mentions) Ab110128, by Abcam (2 mentions) Ab6581, by Abcam (2 mentions) Ab6577, by Abcam (2 mentions) Ab6571, by Abcam (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Milliplex map technology, by Merck Group (1 mentions) Ab2712, by Abcam (1 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Tissuefaxs imaging system, by TissueGnostics (1 mentions) Streptavidin dylight488, by Thermo Fisher Scientific (1 mentions) Clone c8 144b, by Agilent Technologies (1 mentions) A5228, by Novus Biologicals (1 mentions) C digit blot scanner, by LI COR (1 mentions) Anti actin, by Merck Group (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Image studio software, by LI COR (1 mentions) Actin, by Merck Group (1 mentions) Sigma a5228, by Merck Group (1 mentions) Confocal microscope, by Leica camera (1 mentions)

10 protocols using ab6584

Histochemical Analysis of Ocular Tissues

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Eyes were fixed for 48 hours in 4% (w/v) paraformaldehyde solution, rinsed in PBS, and stored overnight in 3% (w/v) sucrose in PBS. Deparaffinized sections of 7 μm thickness were stained with either hematoxylin and eosin or antibodies specific for pan-laminin (NB600-680, Novus), fibronectin (ab6584, Abcam), collagen IV (1340-08, Sanbio), or Iba-1 (019-19741, Wako). Vitreous detection of CCL-2 was performed using a custom Mesoscale Discovery ELISA assay. Detection of the remaining cytokines was performed using MILLIPLEX® MAP technology (Millipore). Four untreated eyes as well as two lipopolysaccharides treated eyes (100 ng/eye) were included as controls [25 (link)]. Statistical differences were evaluated using the Mann-Whitney U test.

Jonckx B., Porcu M., Candi A., Etienne I., Barbeaux P, & Feyen J.H. (2017). Assessment of Ocriplasmin Effects on the Vitreoretinal Compartment in Porcine and Human Model Systems. Journal of Ophthalmology, 2017, 2060765.

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Multicolor Immunofluorescence Staining Protocol

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Following de-paraffinization, antigen retrieval and blocking of peroxidase activity, whole slides were stained overnight at 4°C with primary antibody against TIA-1 (1:50, ab2712, Abcam). Sections were subsequently incubated with anti-mouse Envision+ reagent (K4000, DAKO) for 30 minutes and HRP visualized using cyanine 5 tyramide signal amplification (TSA) according to the manufacturer’s instructions (PerkinElmer). Next, whole slides were stained overnight with primary antibody against CD8 (1:25, clone C8/144B, DAKO) and a biotinylated antibody against fibronectin (1:50, ab6584, Abcam). Slides were incubated with GaM-AF555 (1:150 Life Technologies) and streptavidin-dylight488 (1:150, Thermo Scientific), counterstained with DAPI (Life Technologies) and mounted in prolong gold mounting medium (Life Technologies). Immunofluorescent slides were scanned using a TissueFaxs imaging system (TissueGnostics, Austria). Processed channels were merged using Adobe Photoshop CS5 (Adobe).

van Gool I.C., Eggink F.A., Freeman-Mills L., Stelloo E., Marchi E., de Bruyn M., Palles C., Nout R.A., de Kroon C.D., Osse E.M., Klenerman P., Creutzberg C.L., Tomlinson I.P., Smit V.T., Nijman H.W., Bosse T, & Church D.N. (2015). POLE proofreading mutations elicit an anti-tumor immune response in endometrial cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(14), 3347-3355.

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Immunofluorescence Staining of Key Fibrotic Markers

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Sections were stained for αSMA (1:200, Sigma A5228), collagen type I (1:200, Novus NB600-408) afibronectin (1:500, Abcam ab6584), and calponin (1:200, Abcam ab110128). All samples were blocked with 5% normal donkey serum for 2 hr before incubation in the primary antibody overnight. Samples were then stained with a Cy3 fluorescent-labeled secondary antibody (Jackson Immuno Lab) at 1:200 dilution and nuclear Hoechst stain (1:10000).

Weidenhamer N.K., Moore D.L., Lobo F.L., Klair N.T, & Tranquillo R.T. (2015). Influence of culture conditions and extracellular matrix alignment on human mesenchymal stem cells invasion into decellularized engineered tissues. Journal of Tissue Engineering and Regenerative Medicine, 9(5), 605-618.

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FN Matrix Assembly and Quantification

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FN matrix assembly using deoxycholate (DOC) differential solubilization protocol and Western blot analysis was performed as described previously [48 (link)]. DOC insoluble lysates probed with a polyclonal anti-FN antibody (ab6584, Abcam, Cambridge, MA, USA) under reducing conditions resolved a 220-kDa band of FN. Soluble lysates from the same samples were probed for either CALR (anti-calreticulin polyclonal antibody cat#06-661, EMD Millipore Corporation, Temecula, CA, USA) and or α5 integrin (Ab1928, Abcam, Cambridge, MA, USA). GAPDH (anti-GAPDH mouse monoclonal antibody, Cat#AM4300, Life Technologies, Waltham, MA, USA) or Actin (anti-Actin, Sigma-Aldrich, St. Louis, MO, USA) were used as loading controls. Western Blot detection was done using Western Bright ECL reagent (BioExpress, Kaysville, UT, USA). Images were captured using a C-Digit blot scanner, and imaging data was analyzed by Image studio software (LI-COR Biosciences, Lincoln, NE, USA).

Nair M., Romero J., Mahtabfar A., Meleis A.M., Foty R.A, & Corbett S.A. (2018). Dexamethasone-Mediated Upregulation of Calreticulin Inhibits Primary Human Glioblastoma Dispersal Ex Vivo. International Journal of Molecular Sciences, 19(2), 572.

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Immunohistochemistry of Cytoskeletal Proteins

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Sections were stained for αSMA (1:200; Sigma A5228), collagen type I (1:200; Novus NB600‐408) fibronectin (1:500; Abcam ab6584), and calponin (1:200; Abcam ab110128). All samples were blocked with 5% normal donkey serum for 2 h before incubation in the primary antibody overnight. The samples were then stained with a Cy3 fluorescent‐labelled secondary antibody (Jackson Immuno Lab) at 1:200 dilution and nuclear Hoechst stain (1:10 000).

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Extracellular Matrix Evaluation in Testes

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To evaluate the retention of collagen type I and IV, laminin and fibronectin, the decellularized and intact testes were fixed in 4% paraformaldehyde for 24 h followed by immersing in 30% sucrose as a cryoprotectant for 72 h; at the end, they were transported to the liquid nitrogen. The tissues were then embedded in OCT and sectioned at a thickness of 6–7 μm. Endogenous peroxidase was neutralized by 0.3% H₂O₂ in methanol, and non-specific binding sites were blocked by 4% goat serum in PBS. The samples were incubated with anti-collagen IV (pre-diluted; ab6581, Abcam), anti-collagen I (pre-diluted; ab6577, Abcam), anti-laminin (pre-diluted; ab6571, Abcam), and anti-fibronectin (pre-diluted; ab6584, Abcam) antibodies overnight at 4 °C. Then, they were washed with PBS three times for 15 min. Finally, the samples were treated with streptavidin–horseradish peroxidase complex followed by incubation with diaminobenzidine (DAB, Dako) and counterstained with hematoxylin.

Kargar-Abarghouei E., Vojdani Z., Hassanpour A., Alaee S, & Talaei-Khozani T. (2018). Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells. Stem Cell Research & Therapy, 9, 324.

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Histological Analysis of TPAM Biomaterial

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Histological procedures have been described previously in detail.¹⁶ Briefly, three different lots of TPAM were rehydrated in phosphate buffered saline (PBS) before histology processing. TPAM was then embedded in paraffin and sectioned. Sections were processed with hematoxylin and eosin (H&E, S3301; Dako) and Alcian Blue (Anatech) stains. Sections were processed for immunostaining of fibronectin (Rabbit polyclonal, ab6584; Abcam), collagen type I (Rabbit polyclonal, 2150-0020; Bio-Rad), collagen type III (Rabbit polyclonal, 2150-0100; Bio-Rad), or laminin (Rabbit polyclonal, Z0097; Agilent). Representative images were taken at 40 × with a Confocal Microscope (Leica).

Bonvallet P.P., Damaraju S.M., Modi H.N., Stefanelli V.L., Lin Q., Saini S, & Gandhi A. (2021). Biophysical Characterization of a Novel Tri-Layer Placental Allograft Membrane. Advances in Wound Care, 11(2), 43-55.

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Immunofluorescence Analysis of Fibronectin in Breast Cancer

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Immunofluorescence was performed on representative BCa and adjacent normal tissue from the VUMC cohort. Sections (5µm) were deparaffinized and rehydrated. Antigen retrieval was performed in a steamer either for 30 minutes in pre-warmed modified citrate retrieval buffer (pH 6.0; S2369; Dako) for the total FN or for 12 minutes in Tris-EDTA (TE) buffer (pH 8.0) for ED-A FN. Sections were then blocked in 20% Aquablock/PBS (East Coast Biologics) plus 0.05% Tween-20. The sections being stained for total FN were also treated with an avidin-biotin blocking kit (SP-2001; Vector Laboratories) prior to addition of primary antibody. Primary antibodies were biotinylated rabbit anti-FN (ab6584; Abcam; 1:50) and mouse anti-FN [ED-A] (Clone FN-3E2; 1:200). Detection antibodies were Texas Red conjugated Avidin D (A-1100, Vector Laboratories) and Alexa-647 conjugated goat anti-mouse (Life Technologies). Hoechst 33342 was added to the final incubation step to mark nuclei. Slides were mounted in ProLong Gold Antifade (Invitrogen) and imaged on a BX61 Olympus microscope equipped with a digital camera (Orca ER, Hammamatsu) and controlled with Volocity image acquisition software (PerkinElmer).

Arnold S.A., Loomans H.A., Ketova T., Andl C.D., Clark P.E, & Zijlstra A. (2015). Urinary oncofetal ED-A fibronectin correlates with poor prognosis in patients with bladder cancer. Clinical & experimental metastasis, 33(1), 29-44.

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Immunohistochemistry of Extracellular Matrix Proteins

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Anti-Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti-Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100-immunodepletion), Anti-Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti-Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti-MBP antibody (12 ) (Millipore; rat monoclonal, 1:500), Mouse anti-A2B5 (Invitrogen, Carlsbad, CA; 3 µg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti-GFAP (Millipore, 1:400), Beta-actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000). All immunostaining was visualized using Alexa-fluorophore-conjugated secondary antisera (1:500; Invitrogen).

Sutter P.A., Willis C.M., Menoret A., Nicaise A.M., Sacino A., Sikkema A.H., Jellison E.R., Win K.K., Han D.K., Church W., Baron W., Vella A.T, & Crocker S.J. (2024). Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses. Proceedings of the National Academy of Sciences of the United States of America, 121(5), e2306816121.

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Extracellular Matrix Protein Preservation

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Preservation assessment of collagen types I and IV, fibronectin, and laminin was performed by immunohistochemistry. Briefly, fresh unfixed frozen sections were prepared at 7 μm thickness. Quenching of endogenous peroxidase activity was done by 3% H₂O₂ diluted in methanol for 20 min. Nonspecific binding sites were blocked with 300 μL PBS containing 10% goat serum and 5% bovine serum albumin (BSA) for 1 hour at room temperature and, then, the sections were incubated with biotinylated anti-collagen type I (1:250; ab6577), anti-collagen type IV (1:500; ab6581), anti-fibronectin (1:250; ab6584) and anti-laminin (1:100; ab6571) antibodies overnight at 4 °C (All from Abcam PLC, Cambridge, MA, USA). After washing, 200–400 μL streptavidin-HRP (1:10000; Abcam, USA; ab7403) was added to each section and incubated for 20–30 min at room temperature. Eventually, DAB + chromogen-substrate system (1:50; Dako, Glostrup, Denmark; K3467) and hematoxylin counterstaining were used for detection. A matched similar protocol was done except using primary antibodies incubation as a technical negative control.

Hassanpour A., Talaei-Khozani T., Kargar-Abarghouei E., Razban V, & Vojdani Z. (2018). Decellularized human ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated protocol, as a natural three-dimensional scaffold for construction of bioengineered ovaries. Stem Cell Research & Therapy, 9, 252.

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