The immunofluorescence study of LC3 was performed as previously described[16 (link),19 (link),20 (link)]. Briefly, cultured cells after treatment and infection were washed, fixed and permeabilized and then incubated with rabbit anti-LC3B (Cell Signaling Technology). Secondary antibody was goat anti-rabbit IgG conjugated with Alexa Fluor 594 fluorochrome (Invitrogen Molecular Probes, Eugene, OR, United States). After extensive washing, the nucleic acid stain 4,6-diamidino-2-phenyindole dihydrochloride (DAPI) was added to visualize the nucleus. Then, the coverslips were examined under immunoflourescence conditions by using a Zeiss Axio Observer Z1 (Carl Zeiss, Jena, Germany). The percentage of cells with endogenous LC3 punctate was determined by counting the number of positively stained cells from 100 randomly chosen cells from three separate experiments.
Protocol detail
Immunofluorescence Analysis of LC3
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After treatment
Alexa 594
Anti igg
Antibody
Cells
Cultured cells
Dapi
Fluorochrome
Goat
Immunofluorescence
Infection
Molecular probes
Nucleic acid
Nucleus
Rabbit
Corresponding Organization : Kaohsiung Chang Gung Memorial Hospital
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Variable analysis
independent variables
- Treatment
- Infection
- Percentage of cells with endogenous LC3 punctate
- Cultured cells
- Washing
- Fixation
- Permeabilization
- Incubation with primary antibody (rabbit anti-LC3B)
- Incubation with secondary antibody (goat anti-rabbit IgG conjugated with Alexa Fluor 594 fluorochrome)
- Nuclear staining with DAPI
- Immunofluorescence microscopy
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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