Immunofluorescence of Cultured Cells

doi:10.1038/protex.2015.021

Method Article

Immunofluorescence of Cultured Cells

https://doi.org/10.1038/protex.2015.021

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

Cell biology

Biological techniques

immunofluorescence

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

Humidity Chamber

Wash cells with PBS.
Fix in 4% PFA for 15 min at room temperature (RT).
Wash cells with PBS (2 x 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 x 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 x 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 x 5 min) at RT.
Add mounting media with DAPI and apply coverslip.
Store slides in the dark at 4C.

None.

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Immunofluorescence of Cultured Cells

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