Igg alexa fluor 488

Cells were cultured on coverslips and incubated in DMEM containing 100 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C in the dark. After staining, cells were washed with fresh growth medium, and fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 min. Cells were then permeabilized with PBS containing 0.2% Triton X-100 for 3 min at room temperature, and incubated overnight with anti-HSP60 (1:300; Santa Cruz Biotechnology) or anti-phospho-ERK (Thr202/Tyr204) (1:250; Cell Signaling Technology) antibodies at 4 °C in a darkroom. Next, cells were washed with PBS and incubated with a fluorescently labeled secondary antibody either IgG-Alexa Fluor 594 or IgG-Alexa Fluor 488 (1:300; Cell Signaling Technology) for 1 h at room temperature in the dark. A final incubation was performed to stain the cells with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Jiangsu, China) for 15 min at room temperature. After mounting the coverslips, images were captured using a confocal laser microscope at a magnification of ×600 (Nikon, Telford, UK).

The immunofluorescent staining of cell nuclei, EndoG, mitochondria, and γH2AX foci was performed according the following protocol: cells grown on cover slips were washed in PBS and fixed in 4% paraformaldehyde (in 1xPBS, pH 7.4) for 15 min at room temperature. After washing with PBS, cells were permeabilized using 0.1% Triton-X 100 (in 1xPBS, pH 7.4) at room temperature and then incubated with the blocking reagent (5% Bovine serum albumin in 1xPBS, pH 7.4) for 45 min. The primary antibody: EndoG (ab9647, Abcam), mitochondria antibody [MTC02] (ab3298, Abcam), and γH2AX (ab26350, Abcam) was diluted in 1% bovine serum albumin, (in1xPBS pH 7.4) and incubated with the cells at room temperature. After the incubation, cells were washed with PBS and the fluorescent-labelled secondary antibody diluted in the same buffer was added to cells (IgG-Alexa Fluor 555, A-31572 Invitrogen; IgG-Alexa Fluor 488, #4408, Cell signalling). The cells were incubated for 1 hour in the dark at room temperature. After washing, the DNA was stained with 49-6-diamidine-2-phenyl indole (DAPI, Invitrogen) diluted to a final concentration 1μg/ml in the same buffer. Cells were then washed in PBS and mounted with the anti-fade medium (Vectashield).