Aug 29, 2024
Immunofluorescence and confocal microscopy
- Elias Adriaenssens1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
External link: https://doi.org/10.1038/s41556-025-01712-y
Protocol Citation: Elias Adriaenssens 2024. Immunofluorescence and confocal microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8p1olmk/v1
Manuscript citation:
Adriaenssens, E., Schaar, S., Cook, A.S.I. et al. Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery. Nat Cell Biol 27, 1272–1287 (2025). https://doi.org/10.1038/s41556-025-01712-y
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 03, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103082
Keywords: ASAPCRN, confocal microscopy this protocol detail, confocal microscopy, process of immunofluorescence, immunofluorescence
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the process of immunofluorescence and confocal microscopy.
Materials
28906, Thermo Fisher ScientificPierce™ 16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
0.1% (v/v) Triton X-100 (9002-93-1, Sigma-Aldrich)
5% (v/v) BSA (9048-46-8, Sigma-Aldrich)Bovine Serum Albumin [BSA]Fisher ScientificCatalog #9048-46-8
DAPI Fluoromount-G mounting medium (0100-20, Southern Biotech)
Troubleshooting
Steps
2h 15m
Seed the cells on glass coverslips (12 mm #1.5) at a concentration of 100.000 cells/well, and after treatment with Rapalog for the indicated time, fix in 4% paraformaldehyde (28906, Thermo Fisher Scientific) for 00:10:00 at Room temperature .
10m
After washing with PBS, permeabilize the cells with 0.1% (v/v) Triton X-100 (9002-93-1, Sigma-Aldrich) in PBS for 00:05:00 .
5m
Perform the blocking with blocking buffer (5% (v/v) BSA (9048-46-8, Sigma-Aldrich) and 0.05% (v/v) Triton X-100 diluted in PBS) for 01:00:00 at Room temperature .
1h
Dilute the primary and secondary antibodies in blocking buffer and incubate for 01:00:00 at Room temperature with three PBS washing steps in between.
1h
Mount the cells on microscopy slides in DAPI Fluoromount-G mounting medium (0100-20, Southern Biotech), which stains the nuclei, and store at 4 °C until use.
Perform the confocal microscopy with a Zeiss LSM700 laser scanning confocal microscopy with Plan-Apochromat 40×/1.30 Oil DIC, WD 0.21 mm objective.