A549 tumor xenograft experiments were carried out as previously described [33 (link)]. At a tumor volume of 200-250 mm3, tumors were sham-irradiated or irradiated using a customized shielding device with 1 or 2 × 10 Gy, respectively, using an Xstrahl 200 kV X-ray unit at 1 Gy/min. IR-treated animals were sacrificed 24 h after the last fraction of IR-treatment or 48 hours after the last fraction of high dose IR-treatment. Blood serum was derived from heart-punctured animals following euthanasia. For immunohistochemistry tumor xenografts were fixed in 4% PBS-buffered formalin and embedded in paraffin. Sections (3 mm) were mounted on glass slides, deparaffinized, rehydrated and stained with H&E or a LOX-specific antibody (Novus Biologicals, NB100-2527). Whole tumor sections were quantified for specific LOX-staining intensities. Each treatment group consisted of 3 animals and at least 3 sections per tumor were analyzed. All in vivo experiments were performed in strict accordance with the guidelines for the welfare and use of animals of the Swiss Cantonal Veterinary Authorities and approved by the same Authorities (Permit Number: 145/2011).
Nb100 2527
Manufactured by Novus Biologicals
Sourced in United States
NB100-2527 is a laboratory reagent used for Western blot analysis. It is a monoclonal antibody that specifically binds to the target protein of interest, enabling its detection and quantification in protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab214821, by Abcam (2 mentions)
Ab124762, by Abcam (2 mentions)
Na931, by Cytiva (2 mentions)
Sc 137164, by Santa Cruz Biotechnology (2 mentions)
Na934, by Cytiva (2 mentions)
A5316, by Merck Group (2 mentions)
I9018, by Merck Group (2 mentions)
Hyperpage prestained protein marker, by Meridian Bioscience (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Immobilon, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Human angiogenesis array, by R&D Systems (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Pannoramic 250 flash digital microscope, by 3DHISTECH (1 mentions)
Anti phospho smad1, by Cell Signaling Technology (1 mentions)
9 protocols using nb100 2527
1
A549 Tumor Xenograft Radiation Protocol
2
Immunohistochemical Analysis of Lysyl Oxidase
3
Western Blotting for Protein Expression
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Hearts were homogenized in ice-cold 50 mM HEPES (pH 7.5) containing 100 mM NaCl, 0.5 mM EGTA, 100 mM glycerol-2-phosphate, 10% glycerol, 0.1% Tween 20, supplemented with 1 mM DTT and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were resolved under reducing conditions by sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (Immobilon, Merck-Millipore; Burlington MA, USA, IPVH00010). Membranes were incubated with antibodies against LOX (NB-100-2527, Novus Biologicals, Minneapolis, MN, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374, Merck Millipore). Detection was carried out using suitable horseradish peroxidase-conjugated secondary antibodies (Dako Products, Agilent, Santa Clara, CA, USA) and the LuminataTM Western HRP Substrate (Immobilon, Merck-Millipore). Protein size was assessed using protein molecular weight standards (Hyperpage Prestained Protein Marker; Bioline, Paris, France). Results were normalized by GAPDH protein levels.
4
Quantifying Secreted LOX and Angiogenesis
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LOX secretion was determined by Western blotting performed on concentrated CM samples using an antibody against LOX (NB100-2527, Novus Biologicals). Angiogenesis array on conditioned media was performed using the Human Angiogenesis array (R&D systems) according to the manufacturer`s protocol. For ELISA, cells derived from different tumor entities were sham-treated or irradiated and conditioned media was harvested 24 h after treatment. LOX protein levels were measured according to the protocol of the commercially available LOX-ELISA-kit (USCN Life Science Inc.). Simultaneous quantification of the number of viable cells was performed to allow correction of LOX -levels for cell number.
5
Whole-Cell Lysate Protein Analysis in 2D and 3D Cultures
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Whole-cell lysates from cells in monolayer culture and 3D organoids were prepared as described previously.17 (link) Equivalent amounts (20–40 μg) of protein were loaded into a NuPAGE 4% to 12% Bis-Tris gel. After electrophoresis, transfer to a polyvinylidene difluoride membrane, and blocking with 5% bovine serum albumin or nonfat milk, membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: anti-LOX (1:500, NB100-2527; Novus Biologicals, Centennial, CO), anti-TP63 (1:1000, ab124762; Abcam, Cambridge, UK), anti-IVL (1:1000, I9018; Millipore Sigma, Burlington, MA), anti-DSG1 (1:1000, sc-137164; Santa Cruz Biotechnology, Dallas, TX), anti-BMP2 (1:1000, ab214821; Abcam), anti–phosphorylated-SMAD1/5/9 (1:1000, 13820S; Cell Signaling Technology, Danvers, MA), and anti–β-actin (1:5000, A5316; Millipore Sigma). Immunoblots were detected with an appropriate horseradish peroxidase–conjugated secondary antibody (1:2000, NA934 or NA 931; Amersham BioSciences, Buckinghamshire, UK) by ECL detection (Bio-Rad Laboratories, Hercules, CA). β-actin served as a loading control.
6
Localization and Quantification of Collagen Fibers in Rat Prostate Tissues
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Rat prostate tissues were stained for LOX using the automatic staining system Ventana Benchmark Ultra (Ventana Medical Systems Inc.). Briefly, 4-μm-thick paraffin sections were pretreated with CC1 for antigen retrieval and stained with primary polyclonal antibodies to LOX (NB100-2527, diluted 1:100; Novus Biologicals). Samples were visualized using the ultraView Universal DAB Detection Kit. Sections were analysed using Pannoramic Viewer software. To visualize collagen fibres, paraffin sections from AT-1 tumours at day 7 (n = 5) and 10 (n = 7), and tumour-free controls (n = 5) were stained with Sirius red and examined by polarization microscopy56 (link). The density of Sirius red stained fibres was assessed in 3 photos/animal/group and quantified using Photoshop software (Adobe Photoshop CS5).
7
Immunohistochemical Evaluation of LOX Family
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Protein expression of LOX family members was detected using immunohistochemistry assays with antibodies against LOX (NB100-2527, 1:300, Novus Biologicals, Littleton, CO, USA), LOXL1 (sc-66949, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), LOXL2 (sc-48723, 1:100, Santa Cruz Biotechnology), LOXL3 (37906, 1:300, US Biological, Swampscott, MA, USA), and LOXL4 (ALX-215-067-R050, 1:100, Enzo Life Sciences, Farmingdale, NY, USA). Collagen expression was evaluated by Masson’s trichrome staining. The experiments were repeated with at least three different samples from each group. Image acquisition was performed using a Pannoramic 250 Flash digital microscope (3DHISTECH, Budapest, Hungary).
8
Protein Expression Analysis of Monolayer and 3D Organoids
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Whole-cell lysates from cells in monolayer culture and 3D organoids were prepared as described previously (17 (link)). Equivalent amounts (20–40 μg) of protein were loaded into a NuPAGE 4 to 12% Bis-Tris gel. Following electrophoresis, transfer to a polyvinylidene difluoride membrane, and blocking with 5% bovine serum albumin or non-fat milk, membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were follows: anti-LOX (1:500; NB100–2527; Novus Biologicals, Centennial, CO, USA), anti-TP63 (1:1000; ab124762; Abcam, Cambridge, UK), anti-IVL (1:1000; I9018; Millipore Sigma, Burlington, MA, USA), anti-DSG1 (1:1000; sc-137164; Santa Cruz Biotechnology, Dallas, TX, USA), anti-BMP2 (1:1000; ab214821; Abcam), anti-phospho-SMAD1/5/9 (1:1000;13820S; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:5000; A5316; Millipore Sigma). Immunoblots were detected with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; NA934 or NA 931; Amersham BioSciences, Buckinghamshire, UK) by ECL detection (Bio-Rad Laboratories, Hercules, CA, USA). β-Actin served as a loading control.
9
Immunohistochemistry of LOX Protein in Liver
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