Map2 antibody

Manufactured by Merck Group

Sourced in United States

The MAP2 antibody is a laboratory reagent used in various research applications. It recognizes the microtubule-associated protein 2 (MAP2), which is a structural component of neurons. The MAP2 antibody can be used to detect and visualize MAP2 in biological samples through techniques such as immunohistochemistry and Western blotting.

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Anti-MAP2 antibody (27 citations) Mouse MAP2 antibody (4 citations) Rabbit anti-MAP2 antibody (4 citations) Mouse anti-MAP2 antibody (3 citations) Primary antibody for Map2 (3 citations) Mouse monoclonal antibody against MAP2 (2 citations) MAP2) mouse monoclonal antibody (2 citations) Mouse monoclonal anti-MAP2 antibody (2 citations)

20 protocols using map2 antibody

Rat Hippocampal Neuron Transfection and Imaging

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Rat hippocampal neurons were prepared as previously described (Fink et al., 2003 (link)). Neurons were transfected using
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's
protocol. For live imaging, neurons were plated in chambers (Lab-Tek 155383; Thermo,
Rockford, IL) using NBM (Neurobasal Medium, Gibco [Life Technologies, Carlsbad, CA],
21103-049), supplemented with SM1 (StemCell Technologies [Vancouver, Canada], 05711),
Pen/Strep (Gibco, 15070-063) and 20 mM HEPES (Gibco, 15630). For immunostaining,
cells were fixed in PBS (Gibco, 10010-023) containing 4% Formaldehyde (Ted Pella,
18505) and 120 mM sucrose, stained, and imaged. ArhGAP44 antibody was from Sigma
(1:150, HPA038814), MAP2 antibody was from Chemicon (1:1000, AB 5622; Chemicon, EMD
Millipore, Billerica, MA), pMLC antibody was from Cell Signaling (1:200, 3671S; Cell
Signalling Technology, Danvers, MA), and Rac1 antibody was from Cytoskeleton (1:200,
ARC03; Cytoskeleton, Denver, CO).

Galic M., Tsai F.C., Collins S.R., Matis M., Bandara S, & Meyer T. (2014). Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons. eLife, 3, e03116.

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Quantifying Neuronal Differentiation in PC12 Cells

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Fluorescence immunocytochemistry was conducted for the PC12 cells according to the procedure reported [43 (link),44 (link)]. The cells were fixed on the substrate with 4% paraformaldehyde (PFA, Merck & Co. Inc., Kenilworth, NJ, USA) for 30 min and blocked with 10% goat serum and 0.3% Triton X-100 in PBS for 2 h. The cells were then incubated overnight at 4 °C with rabbit anti-microtubule-associated protein 2 (MAP2) antibody (1:500 dilution; Chemicon). Furthermore, the same cells were incubated with secondary antibodies, DyLight 488 conjugated donkey anti-rabbit (1:250 dilution; Jackson ImmunoResearch) antibody for 1.5 h at 37 °C. The fluorescence images of various fields of the cells that resulted were acquired using fluorescence microscopy (Nikon). Thirty images were recorded for each sample at 20× objective lens and the neurite lengths were analyzed with the Neuron J software [45 (link)] plugin of Image J [46 (link)] to trace the neurite for length measurement.

Chen W.S., Guo L.Y., Tang C.C., Tsai C.K., Huang H.H., Chin T.Y., Yang M.L, & Chen-Yang Y.W. (2018). The Effect of Laminin Surface Modification of Electrospun Silica Nanofiber Substrate on Neuronal Tissue Engineering. Nanomaterials, 8(3), 165.

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Isolation and Culture of Primary Cortical Neurons

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Primary cortical neurons were isolated and cultured as previously reported [22 (link)]. In brief, rat brain cortex tissues were digested using 0.125% trypsin in Hank’s buffer at 37°C for 10 min. The digested cortical tissues were ground. In Neurobasal medium containing 100 U/mL streptomycin, 0.5 mm glutamine, 2% B27, and 100 U/mL penicillin, the isolated monocytes were cultured for 7 days to obtain mature neurons. The obtained neuron purity examined using MAP2 antibody (Chemicon, CA, USA) was over 98%. The phillyrin with different concentrations was used to treat primary cortical neurons for 2 h and then with H₂O₂ of different concentrations to induce neuron injury.

Chen S., Zhang S., Wu H., Zhang D., You G., You J, & Zheng N. (2022). Protective effect of phillyrin against cerebral ischemia/reperfusion injury in rats and oxidative stress-induced cell apoptosis and autophagy in neurons. Bioengineered, 13(3), 7940-7950.

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Stem Cell Differentiation Assay

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The following drugs and reagents were used: lithium chloride (Alfa Aesar B21573, Haverhill, MA, USA), β-catenin antibody (Santa Cruz C2206, Dallas, TX, USA), GSK-3β antibody (Santa Cruz AB15328), Nestin antibody (Abcam ab6142, Cambridgeshire, UK), MAP2 antibody (Merck Millipore AB5622, Burlington, MA, USA), GFAP antibody (Merck Millipore MAB3402C3), neurofilament antibody (Merck Millipore AB15328, MA), DMEM/F12 (Thermo Fisher, Waltham, MA, USA), basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ, USA), epidermal growth factor (EGF, PeproTech), 0.25% trypsin (Sigma Aldrich, St. Louis, MO, USA), 0.01 mol/L RNase (Sigma Aldrich), 0.5 mg/L propidium iodide staining solution (Boster Biology, Wuhan, China), 5% Chloral hydrate (China), and 4% paraformaldehyde (Boster Biology). The equipment included a cold centrifuge (Sigma Aldrich, USA), an inverted light microscope (Olympus IX70, Tokyo, Japan), a microplate reader (Thermo Fisher), an incubator that was set at a temperature of 37°C and regulated with 5% CO₂ (Thermo Fisher), and flow cytometer (FACSCalibur 2, Becton Dickinson, Franklin Lakes, NJ, USA).

Zhang J., He L., Yang Z., Li L, & Cai W. (2018). Lithium chloride promotes proliferation of neural stem cells in vitro, possibly by triggering the Wnt signaling pathway. Animal Cells and Systems, 23(1), 32-41.

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Fluorescence Immunocytochemistry of PC12 Cells

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The fluorescence immunocytochemistry conducted on the PC12 cells was similar to the procedure previously reported [35 (link),36 (link)]. Briefly, the cells were fixed with 4% paraformaldehyde (PFA, Merck & Co. Inc.) for 30 min, then blocked with 10% goat serum and 0.3% Triton X-100 in PBS for 2 h. After overnight incubation at 4 °C with rabbit anti-microtubule-associated protein 2 (MAP2) antibody (1:500 dilution; Merck & Co. Inc.), the cells were washed and further incubated with secondary antibodies, DyLight 488 conjugated donkey anti-rabbit (1:250 dilution; Jackson ImmunoResearch) antibody, for 1.5 h at 37 °C. Twenty-three fluorescence images from various fields were acquired using fluorescence microscopy (Nikon) for each sample using the 20× objective lens. The neurite lengths indicated by the MAP2 staining were analyzed with the Neuron J software (National Institutes of Health, Bethesda, MD, USA) [37 (link)] plugin of Image J [38 (link)].

Chen W.S., Guo L.Y., Masroujeh A.M., Augustine A.M., Tsai C.K., Chin T.Y., Chen-Yang Y.W, & Yang M.L. (2018). A Single-Step Surface Modification of Electrospun Silica Nanofibers Using a Silica Binding Protein Fused with an RGD Motif for Enhanced PC12 Cell Growth and Differentiation. Materials, 11(6), 927.

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Immunofluorescence Analysis of Signaling Proteins

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For immunofluorescence investigations, cells grown on glass coverslips were fixed for 30 minutes at room temperature in 4% paraformaldehyde in 0.1 M phosphate buffer (30) . Cell membranes were permeabilized by incubation for 30 minutes in 0.05 M Tris buffer (pH 7.4) containing 0.1% Triton X-100, 2% BSA, and 2% normal goat serum. Cultures were incubated overnight at 4℃ in primary antibody solution containing either phosphor-APP (Thr668) antibody (1:500, Cell Signaling), MAP2 antibody (1:500, EMD Millipore), p-JNK antibody (1:500, Cell Signaling), JIP-1 antibody (1:50, Santa Cruz), or JIP-3 antibody (1:50, Santa Cruz). Cells were then washed, followed by incubation with fluorescein-isothiocyanate (FITC)- or Cy3-labeled secondary antiserum (1:300, Jackson Laboratories).

Ahn J.H., So S.P., Kim N.Y., Kim H.J., Yoon S.Y, & Kim D.H. (2016). c-Jun N-terminal Kinase (JNK) induces phosphorylation of amyloid precursor protein (APP) at Thr668, in okadaic acid-induced neurodegeneration. BMB Reports, 49(7), 376-381.

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Measuring Neuronal Morphology and Cell Death

Cited 2 times

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Experimenters were blinded to all treatments during analyses. The length of neuronal processes was measured by 2D Sholl analysis using ImageJ as previously described (Bachis et al. 2012 (link)). In brief, cortical neurons were grown on coverslips and fixed as described in immunocytochemistry. Fixed cells were then blocked, permeabilized and incubated overnight at 4 °C with a MAP2 antibody (1:5000; Sigma-Aldrich). Coverslips were washed and then incubated for 1 h at room temperature with the corresponding secondary antibody (1:2000; ThermoFisher Scientific). Cells were imaged with a Zeiss LSM880 microscope as described above. Image scale was calibrated and length of MAP2 positive processes was measured in three randomly selected fields (10 neurons per field) using ImageJ. Experiments were repeated two to four times using different preparations of primary cortical neurons.
Cell death was measured using Hoechst/Propidium Iodide staining (cat#94403, Sigma Aldrich) as previously described (Avdoshina et al. 2016a (link)). Cells were counted with ImageJ.

Wenzel E.D., Bachis A., Avdoshina V., Taraballi F., Tasciotti E, & Mocchetti I. (2017). Endocytic Trafficking of HIV gp120 is Mediated by Dynamin and Plays a Role in gp120 Neurotoxicity. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 12(3), 492-503.

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Immunostaining of neural markers

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The primary antibodies used were as follows: α-synuclein monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA), CNPase, GFAP, MBP, PLP antibodies (Abcam, Cambridge, UK), O4 antibody (Millipore, Darmstadt, Germany), and MAP2 antibody (Sigma, St. Louis, MO, USA).

Yoon Y.S., Ahn W.J., Ricarte D., Ortiz D., Shin C.Y., Lee S.J, & Lee H.J. (2020). Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes. Biomolecules & Therapeutics, 29(1), 83-89.

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Quantifying Neuronal Morphology Using Immunofluorescence

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To determine the length of neuronal processes, neurons were fixed in 4% paraformaldehyde/phosphate buffer with 4% sucrose for 20 min at room temperature. Fixed cells were blocked and permeabilized in 5% non-fat milk in TBS-T (150nM NaCl, 20mM Tris-base, pH 7.5, 0.1% Triton X100) for 1 hr at room temperature. Cells were incubated overnight at 4°C with mouse anti-microtubule associated protein 2 (MAP2) antibody (1:5000; Sigma-Aldrich, MO). Coverslips were washed with PBS-T and corresponding fluorescence-conjugated secondary antibody (1:2000; Invitrogen, CA) were applied for 1 hr at room temperature. Coverslips were washed with TBS-T and mounted with Fluoro-Gel with TES buffer (Electron Microscopy Science, PA). Cells were imaged using an FV300 laser confocal scanning system attached to an Olympus IX-70 (Tokyo, Japan) upright microscope. Image scale was calibrated and length of MAP2 positive processes was measured in three randomly selected fields (10 neurons per field) using ImageJ.

Avdoshina V., Taraballi F., Dedoni S., Corbo C., Paige M., Kont Y.S., Üren A., Tasciotti E, & Mocchetti I. (2016). Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal specific tubulin. Journal of neurochemistry, 137(2), 287-298.

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Immunohistochemical Analysis of 5-HT Neuron Development

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Cells were fixed with 4% paraformaldehyde (PFA), permeabilized, and nonspecific binding sites blocked. To identify 5-HT neurons and analyze their development, a rabbit anti-5-HT antibody was used at a dilution of 1:2000 (Immunostar). The same rabbit anti-5HT antibody was also used in combination with a monoclonal mouse anti-microtubule associated protein 2 (MAP2) antibody (1:2000, Sigma-Aldrich) to distinguish between the axons and dendrites of 5-HT neurons. To study the segregation of serotoninergic and glutamatergic terminals established by 5-HT neurons, the rabbit anti-5-HT antibody was used in combination with a goat anti-SERT antibody (1:1000; C-20, Santa Cruz Biotechnology) and a guinea-pig anti-VGLUT3 antibody (1:3000; Amilhon et al., 2010 (link); Sakae et al., 2015 (link)).

Voisin A.N., Mnie-Filali O., Giguère N., Fortin G.M., Vigneault E., El Mestikawy S., Descarries L, & Trudeau L.É. (2016). Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons. Frontiers in Neuroanatomy, 10, 39.

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