Jun 15, 2023
Immunofluorescent labeling of cultured cells
- 1Northwestern University
- Maryana Nissan: Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
- Divya.D.A Raj: Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Maryana Nissan, Divya.D.A Raj 2023. Immunofluorescent labeling of cultured cells . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjzomlx9/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: June 15, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83495
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
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Abstract
This protocol describes the process of Paraformaldehyde fixation [PFA-fixation] and immunofluorescent-labeling of cultured cells.
Wash cells with 1X Phosphate-Buffered solution [1X PBS].
Fixate cells in 4% PFA for 15 minutes at room temperature [RT].
Wash cells with 1X PBS twice, for 5 minutes each. Here, cells can be stored in 1X PBS at 4C for up to a week.
Permeabilize cells with 1x PBS + 0.1% TriotonX-100 for 5 minutes at RT.
Wash with 1X PBS twice, for 5 minutes each wash at RT.
Block with 10% serum (from species of secondary) for 1 hour at RT.
Drain and incubate with primary antibody diluted in 5% serum overnight at 4C.
Wash in 1X PBS thrice, 5 minutes each wash.
Detect primary antibody with Alexa Fluor secondary antibody diluted 1:400 in 5% serum at RT for 1 hour in the dark.
Wash in 1X PBS thrice, for 5 minutes each wash at RT.
Add mounting media with DAPI and apply the coverslip.
Store slides in the dark at 4C.