Oct 14, 2024

Public workspaceImmunofluorescence of endolysosomal markers in human iNeurons

DOI
dx.doi.org/10.17504/protocols.io.kqdg32zrpv25/v1
  • 1Harvard Medical School;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Frances V Hundley
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.kqdg32zrpv25/v1
Protocol Citation: Frances V Hundley, Miguel A. Gonzalez-Lozano, Harper JW 2024. Immunofluorescence of endolysosomal markers in human iNeurons . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32zrpv25/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: October 02, 2024
Last Modified: October 14, 2024
Protocol Integer ID: 108844
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-025160
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Human induced cortical-like neurons (iNeurons) with endogenously tagged 3xFLAG-EEA1 and TMEM192-3xHA can be used to isolate endosomes and lysosomes, using Endo-IP and Lyso-IP, respectively. Here, we present an immunofluorescence protocol to assess the extent of colocalization between tagged and untagged EEA1 and TMEM192 and markers of the endolysosomal system in iNeurons.

Prepare glass-bottomed dishes & seed iNeurons
Prepare glass-bottomed dishes & seed iNeurons
Coat 24W glass-bottomed dishes with Matrigel.
Seed iNeurons on desired day of differentiation onto Matrigel-coated glass-bottomed dishes.
Fix and stain iNeurons for immunofluorescence
Fix and stain iNeurons for immunofluorescence
1h 10m
1h 10m
Pre-warm 8% paraformaldehyde (PFA) (dilute 16% PFA stock to 8% with PBS) and pre-warm PBS.
Fix iNeurons by adding a volume of 8% PFA equal to the volume of media in the well such that the final concentration of PFA is 4%. Incubate at Temperature37 °C forDuration00:20:00 .

20m
Gently remove fixation solution, and dispose of hazardous waste appropriately. Wash cells once with pre-warmed PBS, and likewise dispose of this hazardous waste wash solution appropriately.
Permabilize cells by adding 0.5% Triton X-100 in PBS at TemperatureRoom temperature for Duration00:20:00 to Duration00:30:00 .

50m
Block cells with blocking solution (3% BSA and 0.1% Triton X-100 in PBS) at TemperatureRoom temperature forDuration01:00:00 .

1h
Gently remove blocking solution and add appropriate primary antibody solution (primary antibody at 1:100-1:200, depending on the antibody in blocking solution). Incubate in primary antibody solution at Temperature4 °C for Duration12:00:00 toDuration16:00:00 .

1d 4h
Gently remove primary antibody solution, and gently wash three times with washing solution (0.02% Tween-20 in PBS), where each wash is at TemperatureRoom temperature for Duration00:05:00 .

5m
Incubate in secondary antibody solution (Alexa Fluor-conjugated secondary antibodies at 1:400 in blocking solution) at TemperatureRoom temperature for Duration01:00:00 .

1h
Gently remove secondary antibody solution and wash once with washing solution.
Stain nuclei with DAPI solution (1ug/mL final concentration DAPI in PBS) at TemperatureRoom temperature for Duration00:10:00 .

10m
Gently removed DAPI solution and gently wash three times with washing solution, where each wash is at TemperatureRoom temperature for Duration00:05:00 .
5m
After final wash, store sample in PBS at Temperature4 °C until micrsocopy analysis.

Spinning disk confocal microscopy
Spinning disk confocal microscopy
Image cells on a confocal microscope using a 100x oil objective.
Data analysis
Data analysis
Use Fiji/ImageJ software to display z-series as maximum intensity projections, then segment images for neuronal soma using CellProfiler Image Analysis Software.
Calculate Mander’s correlation coefficients between two channels with BIOP JACoP plugin in Fiji using the segmented images from the previous step and the Yen thresholding.
Protocol references
Frances V. Hundley, Miguel A. Gonzalez-Lozano, Lena M. Gottschalk, Aslan N. K. Cook, Jiuchun Zhang, Joao A. Paulo, J. Wade Harper. Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons
bioRxiv 2024.09.24.614704; doi: https://doi.org/10.1101/2024.09.24.614704