A series of coronal sections were selected to perform immunochemical staining of BrdU, nestin, DCX, NeuN, and laminin. The primary antibodies were applied including sheep polyclonal anti-BrdU (1 : 500, Abcam, USA), mouse monoclonal anti-BrdU (1 : 1000, Sigma-Aldrich, USA), mouse monoclonal anti-nestin (1 : 1000, Chemicon, USA), mouse monoclonal anti-neuronal nuclei (NeuN, 1 : 1000, Chemicon, USA), or rabbit polyclonal anti-laminin (1 : 200, Sigma-Aldrich, USA). After blocking with goat serum and incubation with primary antibodies overnight at 4°C, the slices were incubated with fluorescent-labeled secondary antibodies Alexa Fluor 488-conjugated anti-sheep IgG (1 : 200), Cy3-conjugated anti-mouse IgG (1 : 200), or Cy3-conjugated anti-rabbit IgG (1 : 200; all three antibodies from Jackson Immunoresearch Laboratories, USA) for 1 h at room temperature. For BrdU immunofluorescence, brain sections were pretreated for 30 min at 37°C with 2 N HCl and then rinsed for 10 min in 0.1 M boric acid (pH = 8.5) at room temperature followed by incubation with blocking solution. Phosphate-buffered saline instead of primary antibody was applied in negative control. The images were taken from four different fields at least (Olympus BX51; Olympus).
Polyclonal rabbit anti laminin
Manufactured by Merck Group
Sourced in United States
Polyclonal rabbit anti-laminin is a laboratory reagent that contains antibodies raised in rabbits against the laminin protein. Laminin is a key component of the extracellular matrix and plays a role in cell adhesion and differentiation. The polyclonal antibodies in this product can be used to detect and study the distribution of laminin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti nestin, by Merck Group (1 mentions)
Cy3 conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Cy3 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Sheep polyclonal anti brdu, by Abcam (1 mentions)
Mouse monoclonal anti brdu, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti α sma, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (1 mentions)
Rabbit monoclonal anti fak, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Ds qi2 camera, by National Instruments (1 mentions)
Opera phenix high content screening system, by PerkinElmer (1 mentions)
Engelbreth holm swarm murine sarcoma basement membrane, by Merck Group (1 mentions)
7 protocols using polyclonal rabbit anti laminin
1
Immunohistochemical Analysis of Neurogenesis
2
Immunohistochemical Evaluation of Angiogenesis
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The following primary antibodies were used: rat monoclonal anti-CD31/Platelet Endothelial Cell Adhesion Molecule-1 (clone: MEC13.3), Catalog No. 550274, BD Pharmingen (San Jose, CA); rabbit polyclonal anti-laminin, Catalog No. L9393, Sigma-Aldrich (St. Louis, MO); mouse monoclonal anti-human nestin (clone: 10C2), Catalog No. MAB5326, Millipore (Billerica, MA); mouse monoclonal anti–α-smooth muscle actin (SMA; clone: 1A4), Catalog No. M0851, Dako (Carpenteria, CA); humanized mouse monoclonal anti–VEGF-A (bevacizumab, Avastin), NDC: 50242-060-01, Genentech (San Francisco, CA); non-specific IgGs, Equitech-Bio Inc. (Kerrville, Tx).
3
Immunodetection of Cell-ECM Interactions
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Primary antibodies: rabbit polyclonal anti-p-FAKTyr397 and anti-tenascin-C (Invitrogen, Carlsbad, CA, USA); mouse monoclonal anti-osteopontin, anti-α-SMA, goat monoclonal anti-Akt1, and rabbit polyclonal anti-vitronectin (Santa Cruz Biotech., Santa Cruz, CA, USA); rabbit polyclonal anti-fibronectin (Dako, Denmark, SC, USA); rabbit monoclonal anti-FAK (Abcam, Cambridge, MA, USA); rabbit polyclonal anti-laminin, mouse monoclonal anti-α-tubulin, and anti-vinculin (Sigma-Aldrich Co., St. Louis, MO, USA); rabbit monoclonal anti-E-cadherin, anti-SMAD2, anti-p-ERK1/2Thr202/204, anti-p-SMAD2Ser465/467, and polyclonal anti-actin, anti-ERK 1/2, anti-SMAD4, and anti-N-cadherin (Cell Signaling Technol., Danvers, MA, USA); mouse monoclonal anti-p-AktSer473, anti-actin, and rabbit monoclonal anti-histone (H3) (Millipore, Billerica, MA, USA). Secondary biotin-conjugated antibodies against rabbit IgG, mouse IgG, and goat IgG (Cell Signaling Technol. Danvers, MA, USA); Streptavidin-conjugated FITC and streptavidin-conjugated horseradish peroxidase (Carlsbad, CA, USA).
4
Muscle Fiber Typing and Size Assessment
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Part of each muscle biopsy sample was frozen in liquid nitrogen-cooled isopentane. These samples were cut into 5-μm thick cryosections using a cryostat at –20 °C. Muscle biopsies were stained for muscle fiber typing and used to assess muscle fiber type composition and type I and II muscle fiber size as described in detail previously [31 (link)]. In short, slides were incubated with primary antibodies directed against myosin heavy chain (MHC)-I (A4.840, dilution 1:25; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and laminin (polyclonal rabbit antilaminin, dilution 1:50; Sigma, Zwijndrecht, the Netherlands). Goat anti-mouse immunoglobulin M (IgM) AlexaFluor555 and goat anti-rabbit immunoglobulin G (IgG) AlexaFluor647 were applied as secondary antibodies (dilution 1:500 and 1:400; Molecular Probes, Invitrogen, Breda, the Netherlands). Images were captured with a fluorescent microscope and analyzed for muscle CSA and fiber-type distribution using ImageJ software.
5
Cryosectioning and Quantification of Megakaryocytes
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Femurs of mice were fixed overnight in 1% paraformaldehyde/phosphate-lysine-sodium periodate and cryoprotected for at least 72 h in a 30% sucrose/phosphate buffer solution at 4°C before subsequent freezing in Sakura Tissue Tek O.C.T. compound (Andwin Scientific). Frozen cryosectioning on slides was performed at the Medical College of Wisconsin Histological Laboratory and Core Center. For MK counts, 7-µm femur sections were rehydrated and permeabilized in TBS-T for 15 min at room temperature (RT) then blocked overnight in a 5% BSA/PBS solution at 4°C. Sections were incubated for 2 h at RT with monoclonal rat-anti-GPIbα (Emfret Analytics) and polyclonal rabbit anti-laminin (Sigma-Aldrich) followed by a 1 h RT incubation with conjugated secondary antibodies (Molecular Probes). Sections were washed and mounted with Prolong Diamond Antifade Mountant with DAPI (Invitrogen) and imaged on a Nikon Eclipse Ti2-E platform equipped with a DS-Qi2 camera and Plan Apo 10x/0.45 (NIS-Elements AR 5.02.00 software). Data were image-processed using Imaris (Bitplane) and Matlab (Mathworks) softwares. Surfaces were created toward the greatest signal intensities GPIbα-positive cells and quantitatively analyzed using Imaris and Excel (Microsoft).
6
Muscle Fiber Type Analysis
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Frozen muscle biopsies were cut into 5-mm-thick cryosections using a cryostat at À20 C, and thaw mounted on uncoated precleaned glass slides. Samples from pre-and postsurgery were mounted together on the same glass slide. Care was taken to properly align the samples for cross-sectional fiber analyses. Muscle biopsies were stained for muscle fiber type determination, that is, type I and type II muscle fibers. Details of the analytical procedures have been described previously. 56 First, antibodies were directed against laminin (polyclonal rabbit anti-laminin, dilution 1:50; Sigma, Zwijndrecht, the Netherlands) and myosin heavy chain-I (A4.840, dilution 1:25;
7
Laminin Binding Assay in FKRP Mutant Cells
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Primary myoblasts and fibroblasts from FKRP P448L mutant and C57BL/6 mice were plated on 384-well imaging plates. Detection of laminin binding to myoblasts and fibroblasts by immunofluorescence was performed as described previously. 22 Myoblasts and fibroblasts were exposed to varying ratios of laminin 111 from Engelbreth-Holm-Swarm murine sarcoma basement membrane (Sigma-Aldrich, St. Louis, MO) and purified recombinant chick mAgrin 21 for 1 hour at 37 C, followed by fixation in 4% paraformaldehyde, and detection with polyclonal rabbit anti-laminin (Sigma-Aldrich) at a dilution of 1:200, and donkey anti-mouse IgG (HþL) Alexa Fluor 488 (Invitrogen, Carlsbad, CA) at a dilution of 1:500. Images were acquired on an Opera Phenix High Content Screening System (PerkinElmer, Waltham, MA). All data are presented as means AE SEM.
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