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    Immunofluorescent staining for neuronal marker MAP2

    Qing Wang

    Published: 2023-04-07 DOI: 10.17504/protocols.io.j8nlkwj46l5r/v1
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    Abstract

    This is the protocol for immunofluorescent staining for neuronal marker MAP2.

    Steps

    1.
    1. Treat differentiated SH-SY5Y cells with 40 ug/mL eumelanin or pheomelanin or PBS for 24 hours.
    2.
    1. Process cells using Cytofix/Cytoperm™ fixation/permeabilization solution (BD554714, Thermo Fisher Scientific) and block with 5% normal goat serum.
    3.
    1. Add primary antibody against microtubule-associated protein 2 (MAP2)(30 µg/mL, OSM00036G, Thermo Fisher Scientific) and incubate at 4℃ overnight.
    4.
    1. Add secondary antibody (1:1000, Alexa 594-conjugated, A11012, Thermo Fisher Scientific) and incubate for 2 hours at room temperature.
    5.
    1. Stain nuclei with DAPI.
    6.
    1. For MAP2-positive cell counting, three images are captured randomly from each well using FluoView FV300 confocal microscope under a 60x objective lens. MAP2 and DAPI channels are merged, and MAP2-positive cells in each random visual field of 0.045000 mm2 are counted using ImageJ.

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