Apr 07, 2023

Public workspaceImmunofluorescent staining for neuronal marker MAP2

DOI
dx.doi.org/10.17504/protocols.io.j8nlkwj46l5r/v1
  • Qing Wang1,2
  • 1Massachusetts General Hospital;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Daniel Choo
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkwj46l5r/v1
Protocol Citation: Qing Wang 2023. Immunofluorescent staining for neuronal marker MAP2. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwj46l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80149
Keywords: ASAPCRN
Funders Acknowledgement:
From cancer associations to altered immunity in the pathogenesis of Parkinson's disease
Grant ID: ASAP-000312
Abstract
This is the protocol for immunofluorescent staining for neuronal marker MAP2.
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.
Protocol references
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