Mouse anti map2

Manufactured by BD

Mouse anti-MAP2 is a monoclonal antibody that specifically binds to Microtubule-associated protein 2 (MAP2), a cytoskeletal protein found in neurons. It is a useful tool for the identification and localization of MAP2 in various research applications.

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Anti-MAP2 (5 citations) Mouse anti (2 citations)

6 protocols using mouse anti map2

Immunocytochemical Analysis of Neural Markers

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Cells were fixed in Accustain (Sigma-Aldrich) for 30 s. Immunocytochemistry was performed using standard pro-tocols. Nuclei were stained with Hoechst 33342 (Invitrogen) and the following primary antibodies: chicken anti-GFAP (1：1,000, Abcam), mouse anti-Nestin (1：500, Chemicon), rabbit anti-Tuj1 (1：500, Covance), rabbit anti-Ki67 (1：500, Novocastra), mouse anti-Map2 (1：500, BD Pharmin-gen) and rabbit anti-NICD (1：500, Abcam). Cells were analysed by conventional epifluorescence microscopy (Leica DM IRE2) and Leica FW4000 software. Image analysis and cell quantification were performed with ImageJ (Wayne Rasband) or Photoshop CS3 (Adobe). A total of 500 to 1,500 Hoechst⁺ cells were counted per coverslip and at least five coverslips were counted per time point and condition.

Herrmann A., Meyer A.K., Braunschweig L., Wagenfuehr L., Markert F., Kolitsch D., Vukicevic V., Hartmann C., Siebert M., Ehrhart-Bornstein M., Hermann A, & Storch A. (2023). Notch Is Not Involved in Physioxia-Mediated Stem Cell Maintenance in Midbrain Neural Stem Cells. International Journal of Stem Cells, 16(3), 293-303.

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Neuronal Culture and Transfection Techniques

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Hippocampal neuronal cultures were prepared from rat embryos at 18 days gestation as previously described [45 (link)]. Neurons were cultured in Neurobasal medium (Life Technologies) supplemented with B27, L-glutamine, penicillin, and streptomycin. Neurons were plated on to glass coverslips coated with poly-D-lysine (0.1mg/ml, Sigma) in a 37°C incubator with 5 % CO2. Neurons were plated at a density of 105,000 cells/cm² for dendrite branching experiments and at a density of 50,000 cells/cm² for spine analysis. Neurons were transfected with pEGFP-C1, pEGFP-C1-Cypin or pEGFP-C1-CypinS using Lipofectamine LTX+PLUS (Invitrogen) following the manufacturer’s protocol for dendrite branching and using calcium phosphate transfection for spine analysis [45 (link)]. For all conditions, neurons were co-transfected with pGW1-mRFP to visualize dendrites and axon. For dendrite branching experiments, neurons were transfected at 7 days in vitro (DIV7) and fixed with 4% paraformaldehyde (PFA) in PBS at DIV12. For spine analysis, neurons were transfected on DIV14 and fixed on DIV17. Cells were immunostained with chicken anti-GFP (1:250; Thermo Fisher Sci.), rabbit anti-RFP (1:250; Rockland) and mouse anti-MAP2 (1:250; BD PharMingen). Immunostaining was visualized with Cy2-, Cy3-, or Cy5-conjugated secondary antibodies (1:500; Thermo Fisher Sci.).

Patel M.V., Swiatkowski P., Kwon M., Rodriguez A.R., Campagno K, & Firestein B.L. (2018). A novel short isoform of cytosolic PSD-95 interactor (cypin) regulates neuronal development. Molecular neurobiology, 55(8), 6269-6281.

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Immuno-Labeling of Glial Progenitor Cells

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GICs were plated at 50,000 cells per milliliter on poly-D-lysine/laminin-coated coverslips in GIC medium with 1 ng/mL EGF and bFGF (Invitrogen). Immunocytochemistry was performed as previously described (Srikanth et al. 2013 (link)) using the following antibodies: rabbit anti-GFAP (1:1000; DakoCytomation) and mouse anti-MAP2 (1:500; BD Pharmingen). Cells were imaged using a Leica SP-5 confocal microscope, and quantification was performed using NIH ImageJ software.

Kouri F.M., Hurley L.A., Daniel W.L., Day E.S., Hua Y., Hao L., Peng C.Y., Merkel T.J., Queisser M.A., Ritner C., Zhang H., James C.D., Sznajder J.I., Chin L., Giljohann D.A., Kessler J.A., Peter M.E., Mirkin C.A, & Stegh A.H. (2015). miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma. Genes & Development, 29(7), 732-745.

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Immunofluorescence Staining of Fixed Brain Sections

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Fixed brains were cut into 30 µm thick sagittal sections using a cryostat. For immunofluorescence (IF) staining, 2-3 brain sections from each mouse were thoroughly washed to remove cryopreservative, blocked in 8% normal horse serum (NHS), diluted in TBS containing 0.1% Triton X-100 for 1 h, and incubated with primary antibodies diluted in PBS containing 2% NHS overnight (1:500 rabbit anti-Iba1, Abcam, ab178846; 1:200 mouse anti-Map2, BD Pharmagen, 556320; 1:500 mouse anti-Gfap, Thermo, 14-9892-82). Following thorough washes and incubation in the appropriate fluorophore-conjugated secondary antibody (1:500, goat anti-mouse Rhodamine-red, Thermo, R6393; or anti-rabbit Rhodamine-red Thermo, R6394) and streptavidin Alexa-flour 488 (1:500, Thermo, S11223) for 1 h at room temperature, sections were mounted on slides with mounting media containing DAPI (Sigma-Aldrich, F6057) for nuclear staining. Representative images of the same regions across all samples were taken using the Keyence BZ-X810 and all image processing was performed using Image J software (FIJI Version 1.51).

Rayaprolu S., Bitarafan S., Santiago J.V., Betarbet R., Sunna S., Cheng L., Xiao H., Nelson R.S., Kumar P., Bagchi P., Duong D.M., Goettemoeller A.M., Oláh V.J., Rowan M., Levey A.I., Wood L.B., Seyfried N.T, & Rangaraju S. (2022). Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain. Nature Communications, 13, 2927.

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Immunofluorescence Staining Protocol

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For immunofluorescence staining, cells were washed twice with PBS without Ca2 + /Mg2 + (LifeTechnologies) and fixed with 4% PFA in PBS for 15 min at RT. PFA was aspirated and cells were washed three times with PBS. Fixed cells were first permeabilized for 10 minutes in 0.2 % Triton X solution and subsequently incubated for 1 hour at RT in blocking solution (5% donkey serum in PBS). Following blocking, primary antibodies were diluted in PBS and cells were incubated with primary antibody solution overnight at 4°C. The following primary antibodies were used: chicken anti-SMI32 (1:10000, Covance), rabbit anti-beta-III-Tubulin (1:3000, Covance), rabbit anti-CHAT (1:1500, Chemicon), mouse anti-MAP2 (1:500, BD Biosciences). Post to the primary antibodies cells were washed three times for 5 min with PBS.
Secondary antibodies were diluted in PBS and incubated with the cells for 1.5 hours at RT. Following secondary antibodies were used: donkey anti-chicken IgY FITC (1:500, Merck Millipore), donkey anti-rabbit IgG647 (1:500, Life Technologies), donkey anti-rabbit IgG488
(1:500, Life Technologies) and donkey anti-mouse IgG555 (1:500, Life Technologies). Nuclei were counter stained using Hoechst (LifeTechnologies).

Kreiter N., Pal A., Lojewski X., Corcia P., Naujock M., Reinhardt P., Sterneckert J., Petri S., Wegner F., Storch A, & Hermann A. (2018). Age-dependent neurodegeneration and organelle transport deficiencies in mutant TDP43 patient-derived neurons are independent of TDP43 aggregation. Neurobiology of disease, 115.

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Immunofluorescent Staining of Transfected Neurons

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Neurons were fixed at DIV 10, approximately 72 h after transfection, in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature (RT). After fixing, coverslips were washed three times in PBS and incubated in blocking buffer (0.1% Triton X-100, 0.02% sodium azide, 2% normal goat serum in PBS) for 1 h at RT. Coverslips were incubated in primary antibodies [chicken anti-GFP (1:500; ThermoFisher, cat. # PA1-9533) and mouse anti-MAP2 (1:1000; BD Biosciences, cat. # 556320)] diluted in blocking buffer overnight at 4°C. After primary antibody incubation, coverslips were washed three times with PBS, after which they were incubated with secondary antibodies [Alexa Fluor 488 goat anti-chicken IgY (1:250; ThermoFisher, cat. # A-11039) and Alexa Fluor 647 donkey anti-mouse IgG (1:250; ThermoFisher, cat. # A-31571)] diluted in blocking buffer for 1 h at RT. Staining for mOrange was not necessary due to inherently high fluorescence. After secondary antibody incubation, coverslips were washed twice with PBS and incubated with Hoechst 33342 for 5 min at RT to stain nuclei. Coverslips were washed one final time with PBS and mounted onto glass microscope slides with Fluoromount G (Southern Biotechnology).

O’Neill K.M., Donohue K.E., Omelchenko A, & Firestein B.L. (2018). The 3′ UTRs of Brain-Derived Neurotrophic Factor Transcripts Differentially Regulate the Dendritic Arbor. Frontiers in Cellular Neuroscience, 12, 60.

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