Mar 21, 2025

Public workspaceConfocal Microscopy of DFP-Treated Cells

DOI
dx.doi.org/10.17504/protocols.io.n2bvj9e9blk5/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj9e9blk5/v1
Protocol Citation: Elias Adriaenssens 2025. Confocal Microscopy of DFP-Treated Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9e9blk5/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: February 05, 2025
Last Modified: March 21, 2025
Protocol Integer ID: 120015
Keywords: ASAPCRN
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 confocal microscopy of DFP-Treated cells after fixation and staining.
Materials
  • ReagentBovine Serum Albumin [BSA]Fisher ScientificCatalog #9048-46-8
  • ReagentDAPI Fluoromount-G®Southern BiotechCatalog #0100-20

Procedure
Procedure
1d 2h 20m
1d 2h 20m
Seed the cells on glass coverslips (12 mm #1.5) at a concentration of 120,000 cells/well, and after treatment with DFP for Duration24:00:00 , fix in 100% methanol for Duration00:20:00 at Temperature-20 °C .

1d 0h 20m
Temperature
After washing with PBS, block the samples with 5% (v/v) BSA (9048-46-8, Sigma-Aldrich) diluted in PBS for Duration01:00:00 at TemperatureRoom temperature .

1h
Incubation
Wash
Temperature
Dilute the primary and secondary antibodies in 5% BSA and incubate for Duration01:00:00 at TemperatureRoom temperature with three PBS washing steps in between.

1h
Incubation
Wash
Temperature
Mount the cells on microscopy slides in DAPI Fluoromount-G mounting medium (0100-20, Southern Biotech), which stains the nuclei, add a cover glass, and store at Temperature4 °C until use.

Temperature
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 or Zeiss LSM900 with Airyscan 2 confocal super-resolution microscopy with Plan-Apochromat 63×/1.4 Oil DIC, WD 0.19 mm objective.
Imaging