Immunofluorescent staining for neuronal marker MAP2
Qing Wang
Published: 2023-04-07 DOI: 10.17504/protocols.io.j8nlkwj46l5r/v1
Abstract
This is the protocol for immunofluorescent staining for neuronal marker MAP2.
Steps
1.
- Treat differentiated SH-SY5Y cells with 40 ug/mL eumelanin or pheomelanin or PBS for 24 hours.
2.
- Process cells using Cytofix/Cytoperm™ fixation/permeabilization solution (BD554714, Thermo Fisher Scientific) and block with 5% normal goat serum.
3.
- Add primary antibody against microtubule-associated protein 2 (MAP2)(30 µg/mL, OSM00036G, Thermo Fisher Scientific) and incubate at 4℃ overnight.
4.
- Add secondary antibody (1:1000, Alexa 594-conjugated, A11012, Thermo Fisher Scientific) and incubate for 2 hours at room temperature.
5.
- Stain nuclei with DAPI.
6.
- 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.
