Authors: Paige Taylor
Abstract
This protocol describes the PFA-fixation and immunofluorescent-labeling of cultured cells.
Reagents
- 4% Paraformaldehyde
- PBS
- PBS + 0.1% TritonX-100
- 10% serum (from species of secondary)
- Primary antibody
- Alexa Fluor 488 secondary
- Alexa Fluor 568 secondary
- DAPI
- Mounting media
Equipment
- Humidity Chamber
Procedure
- Wash cells with PBS.
- Fix in 4% PFA for 15 min at room temperature (RT).
- Wash cells with PBS (2×5 min). Cells can be stored in PBS at 4C for up to a week.
- Permeabilize with PBS + 0.1% TritonX-100 for 5 min at RT.
- Wash with PBS (2×5 min) at RT.
- Block with 10% serum (from species of secondary) for 1 hr at RT.
- Drain and incubate with primary antibody diluted in 5% serum overnight at 4C.
- Wash in PBS (3×5 min) at RT.
- Detect primary antibody with Alexa Fluor secondary antibody diluted 1:400 in 5% serum at RT for 1 hr in the dark.
- Wash in PBS (3×5 min) at RT.
- Add mounting media with DAPI and apply coverslip.
- Store slides in the dark at 4C.
References
Paige Taylor, Krakow Lab
Correspondence to: Paige Taylor ([email protected])
Readers' Comments
Source: Protocol Exchange (2015) doi:10.1038/protex.2015.021. Originally published online 17 March 2015.