Jul 25, 2023

Public workspaceImmunostaining of iPSC-derived neurons for quantification of synaptic proteins

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5x91ng1b/v1
  • Dan Dou1,
  • Erika Holzbaur1
  • 1University of Pennsylvania
Dan Dou
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5x91ng1b/v1
Protocol Citation: Dan Dou, Erika Holzbaur 2023. Immunostaining of iPSC-derived neurons for quantification of synaptic proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x91ng1b/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: July 20, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85305
Keywords: ASAPCRN, culture of neuron, synaptic protein, transfection of ipsc, derived neuron, quantifying somal protein, neurons for quantification, somal proteins of interest, human ipsc, neuron, imaging of axonal cargo, neurons for the purpose, ipsc, immunostaining, axonal cargo
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Materials
35 mm imaging dish, 20 mm glass diameter (Mattek, p35g-1-5-20-c)
18mm circular coverglass (Electron Microscopy Sciences, Cat# 72222-01)
Prolong Gold Antifade Mountant (Thermo Fisher, P36930)
Troubleshooting
At DIV14, fix human iNeurons in 4% paraformaldehyde supplemented with 4% sucrose for 15 minutes at 37 degrees C

Wash four times with PBS
Permeabilize for 15 minutes in 0.2% Triton-X in PBS
Block for 1 hour with 5% goat serum and 1% BSA in PBS
Incubate in primary antibody diluted in blocking solution at room termperature for 1 hour
Wash three times with PBS
Incubate in secondary antibody diluted in blocking solution for 1 hour at room temperature

Wash three times with PBS
Remove PBS and add 40 µL Prolong Gold (Thermo Fisher). Using forceps, apply coverglass.