Jun 26, 2023

Public workspaceImmunocytochemistry of primary neurons

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjdkmlx9/v1
  • giulia.tombesi1
  • 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
giulia.tombesi
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DOI: dx.doi.org/10.17504/protocols.io.eq2lyjdkmlx9/v1
Protocol Citation: giulia.tombesi 2023. Immunocytochemistry of primary neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjdkmlx9/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: June 26, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84047
Keywords: staining, primary neurons, cell culture, immunocytochemistry, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
This protocol describes the steps to obtain a great staining of primary neurons.
Materials
Blocking Solution: 1% BSA , 50mM Glycine , 2% FBS/Goat Normal Serum/ Donkey Normal Serum, 0,1% TRITON X-100 in PBS1X.

Working Solution: Amount10 mL Blocking Solution in Amount40 mL PBS1X

Cells Fixation
Cells Fixation
30m
30m
Wash cells with PBS1X/HBSS
Fix cells with paraformaldehyde (PFA) 4% for Duration00:30:00 at room temperature

30m
Wash cells with PBS1X for Duration00:05:00 for three times at room temperature
5m
Cells permeabilization and saturation
Cells permeabilization and saturation
45m
45m
Permeabilize cells with 0.3% Triton X-100 (in PBS1X) for Duration00:05:00 at room temperature

5m
Wash cells with PBS1X for Duration00:05:00 for three times at room temperature
5m
Saturate cells in blocking solution for at least Duration00:30:00

30m
Wash cells with PBS1X for Duration00:05:00 for three times at room temperature
5m
Incubation with primary antibody
Incubation with primary antibody
1h 35m
1h 35m
Incubate with primary antibody in working solution for Duration01:30:00 at room temperature

1h 30m
Wash cells in working solution for Duration00:05:00 for three times at room temperature
5m
Incubation with secondary antibody
Incubation with secondary antibody
1h 6m
1h 6m
Incubate with secondary antibody in working solution for Duration01:00:00 at room temperature in the dark

1h
Wash cells in working solution for Duration00:05:00 for three times at room temperature
5m
Wash cells in PBS1X for Duration00:01:00 for three times at room temperature
1m
Mounting
Mounting
Rinse coverslips in dH2O
Mount the coverslips on slides with mounting medium