Alexa fluor 594 fluorochrome

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.

Cells were plated directly on a coverslip and allowed to adhere with overnight incubation. The following day, the cells were irradiated and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were then washed with PBS and permeabilized with 70% ethanol overnight at 4°C and for 20 minutes with 0.1% Igepal at room temperature. Cells were washed, blocked with 2% BSA for 1 hour and incubated overnight with 1:1,000 Aurora Kinase A antibody (Cell Signaling Technology). Centrosomes were visualized by 1 hour incubation with a 1:500 AlexaFluor 594 fluorochrome (Invitrogen). Cells were then incubated with 1:500 alpha tubulin (Santa Cruz Biotechnology) for 1 hour at room temperature. Mitotic spindles were visualized by 1 hour incubation with 1:600 FITC (Jackson ImmunoResearch), and DNA was stained with 1 μg/mL DAPI (RRID:AB_2893474; Sigma). Pictures were captured with a Leica microscope.
BRCA1 foci were visualized by incubating 1:500 BRCA1 antibody overnight (Santa Cruz Biotechnology) and 1:600 FITC (Jackson ImmunoResearch) for 45 minutes at room temperature. Foci for at least one hundred cells were manually counted per experiment using ImageJ software. Each foci experiment was repeated at least once.