Mab342

Manufactured by Merck Group

MAB342 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications. The core function of MAB342 is to facilitate precise and controlled experimental procedures. Further details on the intended use or specific applications of this product are not available.

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4 protocols using mab342

NMDA-Induced Changes in GLUT1 and MCT1 Levels

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For GLUT1 and MCT1 immunocytochemistry, differentiated cells were treated for 30 min at 37°C with 100 μM NMDA. Glycine as co-agonist for NMDA receptors was already present in the medium. NMDA receptor blockers D-AP5 and/or 7CKA (100 μM) were applied 30 min prior to NMDA stimulation. Plasma membranes of mature oligodendrocytes were labeled with anti-galactocerebroside (GalC) antibody (Millipore Cat# MAB342 RRID: AB_94857) for 15 min at 37°C prior to fixation with 4% paraformaldehyde for 10 min at RT. Cells were permeabilized with ice-cold methanol for 5 min, washed with PBS, and incubated with anti-GLUT1 antibody (Abcam Cat# ab32551 RRID: AB_732605) or with anti-MCT1 (Millipore Cat# AB1286 RRID: AB_90565) overnight at 4°C. Analysis was carried out on confocal stacks of individual cells using ImageJ (http://rsb.info.nih.gov/ij/). Data are presented as the ratio of GLUT1 or MCT1 signal area to GalC area. Three individual experiments with 14–24 cells per condition were analyzed. Data were grouped per experiment and normalized to control condition.

Saab A.S., Tzvetavona I.D., Trevisiol A., Baltan S., Dibaj P., Kusch K., Möbius W., Goetze B., Jahn H.M., Huang W., Steffens H., Schomburg E.D., Pérez-Samartín A., Pérez-Cerdá F., Bakhtiari D., Matute C., Löwel S., Griesinger C., Hirrlinger J., Kirchhoff F, & Nave K.A. (2016). Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism. Neuron, 91(1), 119-132.

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Immunofluorescence of Cerebellar Cells

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Cells and cerebellar slices were fixed in 4% paraformaldehyde for 10 and 40 min, respectively. Samples were washed with PBS, permeabilized and blocked in 4% normal goat serum, 0.1% Triton X‐100 in PBS (blocking buffer) for 1 h and incubated overnight at 4 °C with primary antibodies against MBP (#SMI 99, 1:1000; Biolegend), rat anti‐O4 (1:50, kindly supplied by Dr. Chistine Thompson, University of Glasgow), GalC (#MAB342, 5 µg/ml; Millipore), neurofilament light polipeptide (NFL, #C28E10, 1:200; Cell Signalling), Olig2 (#MABN50, 1:1000; Millipore) and APC (CC1, #OP80, 1:200, Millipore). Samples were washed in PBS and incubated with fluorophore-conjugated Alexa secondary antibodies (1:200) in blocking buffer for 1 h at room temperatue (RT). Cell nuclei were detected by incubation with DAPI (4 μg/ml, Sigma‐Aldrich). Samples were mounted with Fluoromount‐G.

Quintela-López T., Ortiz-Sanz C., Serrano-Regal M.P., Gaminde-Blasco A., Valero J., Baleriola J., Sánchez-Gómez M.V., Matute C, & Alberdi E. (2019). Aβ oligomers promote oligodendrocyte differentiation and maturation via integrin β1 and Fyn kinase signaling. Cell Death & Disease, 10(6), 445.

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Characterization of Pluripotent Stem Cells

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Di-hydrotashinone (DT, 1 µM, D0947, Sigma-Aldrich); actinomycin-D (1 μg/ml, CID 2019, Sigma-Aldrich); thiazolyl blue tetrazolium bromide – MTT (M2128, Sigma-Aldrich); anti-Nestin (MAB353, Millipore); anti-DCX (sc8066, Santa Cruz); anti-HuR (390,600, Invitrogen); anti-βTubIII (MMS-435P, Covance); anti-MAP2 (Ab5622, Millipore); anti-GFAP (PRB571c, Covance); anti-GalC (Mab 342, Millipore); anti-Olig2 (Ab9610, Millipore); pluripotent stem cells 4-Marker Immunocytochemistry Kit (A24881, Thermo Fisher); 4,6, diamino-2-phenyl-l-indole di-hydrocloride–DAPI (D259542, Sigma-Aldrich), Alexa Fluor® 488 (Life Technologies) Alexa Fluor® 546 (Life Technologies), cresyl-violet (Sigma-Aldrich), Peroxidase AffiniPure Goat Anti-Rabbit/Mouse IgG (Jackson).

Carelli S., Giallongo T., Rey F., Latorre E., Bordoni M., Mazzucchelli S., Gorio M.C., Pansarasa O., Provenzani A., Cereda C, & Di Giulio A.M. (2019). HuR interacts with lincBRN1a and lincBRN1b during neuronal stem cells differentiation. RNA Biology, 16(10), 1471-1485.

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Immunofluorescence Assay for Cell Markers

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For immunofluorescence assays, cells were fixed with 4% paraformaldehyde and 2% sucrose in PBS for 5 min, and then permeabilized with 0.01% Triton X-100 in 4% paraformaldehyde for 5 min. For detection of hBD2 and hBD3, goat anti-hBD2 and goat anti-hBD3 (R&D Systems) were used. HSPGs and GalCer were detected with mouse monoclonal antibodies 10E4 (US Biological) and MAB342 (Millipore), respectively. CCR5 and CXCR4 were immunostained with mouse monoclonal antibodies (both from R&D and provided by the NIH AIDS Reagent Program). For detection of HIV, mouse anti- p24 antibody was used (NIH AIDS Reagent Program). Tight junction protein ZO-1 was detected using rabbit antibody (Invitrogen). Secondary antibodies labeled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate were purchased from Jackson ImmunoResearch. The specificity of each antibody was confirmed by negative staining with the corresponding isotype control antibody. Cell nuclei were counterstained with TO-PRO-3 iodide (blue) or propidium iodide (red) (Molecular Probes). Cells were analyzed by using a krypton-argon laser coupled with a BioRad MRC2400 confocal head. The data were analyzed by using BioRad LaserSharp software.

Herrera R., Morris M., Rosbe K., Feng Z., Weinberg A, & Tugizov S. (2015). Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells. Virology, 487, 172-187.

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