Apr 30, 2025

Public workspaceImmunofluorescence-based Assay for Monitoring pUb in Pink1-Parkin-Mediated Mitophagy in iPSC-Derived Dopaminergic Neurons

DOI
dx.doi.org/10.17504/protocols.io.yxmvm95jol3p/v1
  • 1Montreal Neurological Institute - McGill University
Roxanne Larivière
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm95jol3p/v1
Protocol Citation: Roxanne Larivière, Edward A. Fon 2025. Immunofluorescence-based Assay for Monitoring pUb in Pink1-Parkin-Mediated Mitophagy in iPSC-Derived Dopaminergic Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm95jol3p/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 is working
Created: January 08, 2025
Last Modified: April 30, 2025
Protocol Integer ID: 117936
Keywords: IF, immunofluorescence, MAP2, mitophagy, iPSC, dopaminergic neurons
Funders Acknowledgements:
Michael J. Fox Foundation for Parkinson's research
Grant ID: MJFF-020696
Abstract
This protocol outlines the procedure for assaying pUb in Pink1-Parkin-mediated mitophagy in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons using immunofluorescence.






Materials







Before start
iPSC-derived dopaminergic neurons are cultured on 96-well plates for up to 4 to 6 weeks of differentiation. We use black Costar 96-well assay plates (Corning cat. no. 3904), which are compatible with imaging using the Opera Phenix high-content screening system.

Be extremely gentle when dispensing into the wells, as dopaminergic neurons are prone to detaching with forceful pipetting.
CCCP treatment
CCCP treatment
4h
4h
- Treat neurons (at least 5 columns of one 96 well plate) with CCCP 20µM for Duration04:00:00

4h
Toxic
Fixation of dopaminergic neurons in 96 well plates
Fixation of dopaminergic neurons in 96 well plates
20m
20m
- Leave Amount50 µL of media and gently add Amount50 µL of 8% PFA per well.
- Incubate at room temperature Duration00:20:00 .
- Remove PFA and wash 3 times with 1X PBS, Amount75 µL per well, Duration00:05:00 each

20m
Permeabilization
Permeabilization
25m
25m
- Prepare permeabilization solution: 0.2% TX-100; 1X PBS.
- Incubate in permeabilization solution Duration00:10:00 , Amount60 µL per well
- Remove permeabilization solution and wash 3 times with 1X PBS, Amount75 µL per well, Duration00:05:00 each

25m
Blocking
Blocking
1h
1h
- Prepare blocking buffer: 5% normal goat serum; 0.02% TX-100; 1X PBS
- Incubate in blocking solution Duration01:00:00 , Amount60 µL per well

1h
Incubation with primary antibodies
Incubation with primary antibodies
16h
16h
- Prepare primary antibody mix in blocking buffer, Amount60 µL per well

  • rabbit pUb antibody 1:1250
  • chicken MAP2 antibody 1:1500

- Incubate DurationOvernight at Temperature4 °C with gentle shaking

16h
Washes
Washes
20m
20m
- Wash 4 times withAmount75 µL 1X PBS, Duration00:05:00 each

20m
Incubation with secondary antibodies
Incubation with secondary antibodies
2h
2h
- Prepare secondary antibody mix in 1% normal goat serum; 1X PBS. Dispense Amount60 µL per well.

  • anti-Rabbit Alexa Fluor 488 1:1000
  • anti-Chicken Alexa Fluor 555 1:1000
  • Hoechst 33342 1:5000

- Incubate Duration02:00:00 at TemperatureRoom temperature with gentle shaking

2h
Washes
Washes
20m
20m
- Wash 4 times with Amount75 µL 1X PBS, Duration00:05:00 each
- Leave Amount100 µL of 1X PBS into wells

20m
Imaging
Imaging
- Samples are ready for imaging