Ab128567

HeLa PINK1^−/− cells stably expressing YFP-Parkin and HsPINK1 were seeded on HistoGrip-coated coverslips 48 hours before treatment with OA and/or 200 μM kinetin for the indicated times. Cells were then fixed with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer on a rocker for 10 min, rinsed three times with PBS, and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min. After that, samples were blocked with 3% (v/v) goat serum in 0.1% (v/v) Triton X-100/PBS for 15 min and incubated with anti–green fluorescent protein (Thermo Fisher Scientific, A10262, RRID:AB_2534023) and anti-mitochondrial HSP60 (Abcam, ab128567, RRID:AB_11145464) antibodies for 90 min. Following three washes with PBS and subsequent 1 hour of incubation with Alexa Fluor 488 goat anti-chicken IgG (Thermo Fisher Scientific, A32931, RRID:AB_2762843) and Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, A21235, RRID:AB_2535804), the coverslips were washed three times with PBS and mounted with a tris-buffered DABCO-glycerol mounting medium onto glass slides. Imaging of the coverslips was done with an inverted Leica SP8 confocal laser scanning microscope under 63×/1.40 numerical aperture objective (Oil immersion, HC PLAPO, CS2; Leica microsystems). Details on the staining procedure are available at (49 (link)).