Mar 13, 2025
Immunofluorescence Staining for Assessing pUb(Ser65) Accumulation in iNeurons
- Benjamin O'Callaghan1,2,
- Helene Plun-Favreau1,2
- 1Department of Neurodegenerative Diseases, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA, 20815
Protocol Citation: Benjamin O'Callaghan, Helene Plun-Favreau 2025. Immunofluorescence Staining for Assessing pUb(Ser65) Accumulation in iNeurons. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6okpgx9/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: March 04, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 123771
Keywords: ASAPCRN, pUb(Ser65), PINK1, mitophagy, iNeuron, analysis of ineuron, accumulation in ineurons protocol, dependent mitophagy initiation, ineuron, ineurons protocol, immunofluorescence, immunofluorescence in order, dependent mitophagy
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000478
Abstract
Protocol for the imaging and analysis of iNeurons with anti-pUb(Ser65) immunofluorescence in order to measure PINK1-dependent mitophagy initiation.
Materials
- 10x Phosphate Buffered Saline (Fisher Scientific, 10649743)
- Heat-inactivated foetal bovine serum (FBS) (Gibco, A5256801)
- 10% w/v Triton-X-100 (Sigma, 93443)
- rabbit anti-pUb(Ser65) IgG (Cell Signaling Technology, Cat# 62802, RRID:AB_2799632)
- mouse anti-TOM20 IgG2a (Santa Cruz Biotechnology, Cat# sc-17764, RRID:AB_628381)
- chicken anti-MAP2 IgY (Millipore Cat# AB5543, RRID:AB_571049)
- goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165)
- goat anti-mouse IgG (H+L) Alexa Fluor 568 (Thermo Fisher Scientific Cat# A-11004, RRID:AB_2534072)
- goat anti-chicken IgY (H+L) Alexa Fluor 647 (Thermo Fisher Scientific Cat# A-21449, RRID:AB_2535866).
- Hoechst 33342 Solution (20 mM) (Thermo Fisher Scientific Cat# 62249)
Troubleshooting
Immunofluorescence Staining
4h
iNeuron culture and formaldehyde fixation described in "iNeuron Differentiation and Culture in N2B27 vs BrainPhys for Immunofluorescence and Biochemistry Assessments of Mitophagy" (https://www.protocols.io/private/E5851436F8F111EF86990A58A9FEAC02).
Wash fixed iNeuron cultures in Revvity 96-well Phenoplates twice with 100 µL of PBS.
Note: cultures are very delicate and utmost care and steady liquid additions is required throughout the whole staining procedure in order to prevent detachment of iNeurons from culture dish en masse.
Incubate fixed iNeurons in blocking and permeabilisation solution (10 % (v/v) FBS, 0.25 % (w/v) , 1x PBS) for 01:00:00 at Room temperature . 50 µL per well of Revvity Phenoplate 96-well.
Prepare primary antibody dilution in blocking solution (10 % (v/v) FBS, 1x PBS). 50 µL will be required for each well of a Revvity Phenoplate 96-well.
1:1000 rabbit anti-pUb(Ser65) IgG
1:1000 mouse anti-TOM20 IgG2a
1:5000 chicken anti-MAP2 IgY
Remove blocking and permeabilisation solution from cells and add 50 µL of primary antibody dilution prepared in to each well of fixed iNeurons. Incubate for 02:00:00 at Room temperature .
2h
Remove primary antibody dilution and wash wells 3x with 100 µL of PBS.
Prepare secondary antibody and Hoechst nuclear counterstain dilution in blocking solution (10 % (v/v) FBS, 1x PBS). 50 µL will be required for each well of a Revvity Phenoplate 96-well.
1:2000 goat anti-rabbit IgG (H+L) Alexa Fluor 488
1:2000 goat anti-mouse IgG (H+L) Alexa Fluor 568
1:2000 goat anti-chicken IgY (H+L) Alexa Fluor 647
10 µM Hoechst 33342
Remove PBS wash from cells and add 50 µL of secondary antibody with Hoechst dilution prepared in to each well of fixed iNeurons. Incubate for 02:00:00 at Room temperature .
2h
Remove secondary antibody with hoechst dilution and wash wells 3x with 100 µL of PBS.
Keep cells incubated in 100 µL of PBS for imaging.
Imaging
Imaging Parameters on Opera Phenix:
Use 40x objective
In confocal mode do a z-stack
1µm thick slices
Sequentially detect the 4x fluoroscent signals
Adjust pixel dwell time to give good signal but avoiding saturation
Hoechst 33342: Excitation 375nm laser, Emission 435-515nm
Alexa Fluor 488: Excitation 488nm laser, Emission 500-550nm
Alexa Fluor 568: Excitation 561nm laser, Emission 570-630nm
Alexa Fluor 647: Excitation 640nm laser, Emission 650-760nm
I usually do 6x fields per well and will take ~1.5hr to image 60-wells
Analysis
Using the columbus software the following is a useful analysis strategy for pUb(Ser65) accumulation in iNeurons:
- Using max intensity project
- Define MAP2+ area of each imaging field based on the intensity of Alexa Fluor 647 (MAP2 Secondary antibody)
- Measure Alexa Fluor 488 (pUb(Ser65) secondary antibody) mean intensity in the MAP2+ area of each field.
- Report the mean intensity of all fields within a well.
- Average the mean well intensities across technical replicate wells
Protocol references
Soutar MPM, Melandri D, O'Callaghan B, Annuario E, Monaghan AE, Welsh NJ, D'Sa K, Guelfi S, Zhang D, Pittman A, Trabzuni D, Verboven AHA, Pan KS, Kia DA, Bictash M, Gandhi S, Houlden H, Cookson MR, Kasri NN, Wood NW, Singleton AB, Hardy J, Whiting PJ, Blauwendraat C, Whitworth AJ, Manzoni C, Ryten M, Lewis PA, Plun-Favreau H. Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at 16q11.2 and MAPT H1 loci. Brain. 2022 Dec 19;145(12):4349-4367. doi: 10.1093/brain/awac325. PMID: 36074904; PMCID: PMC9762952.