Immunofluorescence of Cultured Cells

• Protocol
• Discussion

Authors: Paige Taylor

Abstract

This protocol describes the PFA-fixation and immunofluorescent-labeling of cultured cells.

Reagents

1. 4% Paraformaldehyde
2. PBS
3. PBS + 0.1% TritonX-100
4. 10% serum (from species of secondary)
5. Primary antibody
6. Alexa Fluor 488 secondary
7. Alexa Fluor 568 secondary
8. DAPI
9. Mounting media

Equipment

1. Humidity Chamber

Procedure

1. Wash cells with PBS.
2. Fix in 4% PFA for 15 min at room temperature (RT).
3. Wash cells with PBS (2×5 min). Cells can be stored in PBS at 4C for up to a week.
4. Permeabilize with PBS + 0.1% TritonX-100 for 5 min at RT.
5. Wash with PBS (2×5 min) at RT.
6. Block with 10% serum (from species of secondary) for 1 hr at RT.
7. Drain and incubate with primary antibody diluted in 5% serum overnight at 4C.
8. Wash in PBS (3×5 min) at RT.
9. Detect primary antibody with Alexa Fluor secondary antibody diluted 1:400 in 5% serum at RT for 1 hr in the dark.
10. Wash in PBS (3×5 min) at RT.
11. Add mounting media with DAPI and apply coverslip.
12. 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.