May 24, 2023
Immunostaining of iPSC-derived neurons
- Dan Dou1,2,
- Erika L.F. Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, Erika L.F. Holzbaur 2023. Immunostaining of iPSC-derived neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj4kdlmk/v1
Manuscript citation:
Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur EL, Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports 42(5). doi: 10.1016/j.celrep.2023.112448
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: March 10, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 78438
Keywords: ASAPCRN, culture of neuron, transfection of ipsc, derived neuron, axonal abundance of protein, imaging of axonal cargo, human ipsc, neuron, axonal cargo, neurons for the purpose, axonal abundance, ipsc, protein
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Grant ID: ASAP-000350
Abstract
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal and axonal abundance of proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Materials
Bouin's solution (Sigma, HT10132)
BrainPhys Neuronal Medium (Stemcell Technologies, Cat# 05790)
Methanol, Optima LC/MS grade (Thermo Fisher, A456-4)
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
Replace culture media in dish with 1 mL room temperature BrainPhys culture media (Stemcell Technologies)
Add 1 mL Bouin's solution (Sigma). Incubate at room temperature for 30 minutes.
Wash at least 5 times with PBS. Yellow color should be absent.
Permeabilize in ice-cold Optima methanol (Thermo Fisher) at -20 degrees C for 8 minutes.
Wash 3 times in PBS.
Block for one hour (5% goat serum and 1% BSA in PBS).
Incubate in primary antibody in blocking solution for 1 hour at room temperature.
Wash 3 times in PBS.
Incubate in secondary antibody in blocking solution for 1 hour at room temperature.
Wash 3 times in PBS.
Remove PBS and add 40 µL Prolong Gold (Thermo Fisher). Using forceps, apply coverglass.