Immunofluorescence was performed as previously described15 (link) with slight modification. In brief, cells on the coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Cells were incubated with primary antibodies in 5% BSA in PBS supplemented with 0.1% Tween 20 first at room temperature for 30 min, and then at 4 °C for overnight. The next day, cells were probed with Alexa Fluor-conjugated secondary antibody (Life Technologies), washed and mounted with ProLong Gold (Life Technologies). The slides were imaged with Leica TCS SP8 fluorescent confocal microscope. If cells were subjected to DAPI staining, cells were incubated with 1 μg/ml DAPI in PBS for 5 min, and washed with PBS before mounting.
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Immunofluorescence Staining Protocol
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Alexa Fluor-conjugated secondary antibody
ProLong Gold
Leica TCS SP8 fluorescent confocal microscope
Antibodies
Antibody
Cells
Confocal microscope
Dapi
Gold
Immunofluorescence
Paraformaldehyde
Triton x 100
Tween 20
Corresponding Organization :
Other organizations : University of Pennsylvania, UiT The Arctic University of Norway, Children's Hospital of Philadelphia, Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute, University of California, San Francisco
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Variable analysis
independent variables
- Primary antibodies
- Alexa Fluor-conjugated secondary antibody staining
- DAPI staining
- Cells on the coverslips
- 4% paraformaldehyde fixation
- 0.5% Triton X-100 permeabilization
- 5% BSA in PBS supplemented with 0.1% Tween 20
- Incubation time and temperature (room temperature for 30 min, and then at 4 °C for overnight)
- Leica TCS SP8 fluorescent confocal microscope
- Not explicitly mentioned
- Not explicitly mentioned
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