Mar 01, 2022
Immunofluorescent Staining
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. Immunofluorescent Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.b5s5q6g6
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 01, 2022
Last Modified: March 01, 2022
Protocol Integer ID: 58941
Abstract
This protocol is used to stain cryosectioned mouse brain tissue.
To cryo-sectioned brain tissue, wash with 1X phosphate buffered saline (PBS) for 3x 5-minute washes.
15m
Incubate in blocking buffer for 1 hour at room temperature.
Blocking buffer: 10% serum + 0.5% Triton X-100 in 1X PBS
1h
Wash tissue with 1X PBS.
5m
Incubate in primary antibody diluted in blocking buffer overnight at 4oC.
1d
Wash tissue with 1X PBS for 5x 5-minute washes.
30m
Incubate in secondary antibody diluted in blocking buffer for 1 hour at room temperature.
1h
Wash tissue with 1X PBS for 5x 5-minute washes.
30m
If tissue wasn't previously mounted on a slide, mount on a superfrost plus slide and let dry at room temperature for at least 10 minutes.
15m
Coverslip with fluorescent mounting medium and a #1.5 coverslip. Outline the coverslip with clear nailpolish.
1m