Anti mouse igg h l alexa fluor 568

Cells (5 × 10⁴ cells) were grown on a sterilized confocal dish (Coverglass-Bottom Dish, SPL. Cat#100350) for 24 h. Cells were incubated with Mitotracker (Invitrogen, #M22426) for 30 min and fixed with 4% formaldehyde for 30 min. These cells were then washed with PBS twice and incubated with TRIP-Br1 (Enzo Life Sciences, ALX-804-645) or LC3 (Cell Signaling Technology, #2775S) antibodies overnight at 4°C. These primary antibodies were detected with an anti-mouse IgG H&L (Alexa Fluor® 568) (Abcam, #ab175473) or an anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, #ab150077). Nuclei were stained with DAPI (Invitrogen, Cat#P36931) for 10 min after washing with PBS. Colocalization between fluorophores was analyzed using ImageJ software (ver. 1.51u; National Institutes of Health, USA). For lysosomal confocal imaging, cells were incubated with 100 nM Lysotracker (Invitrogen, Cat# L12492) in a phenol-free medium for 90 min. Confocal images were obtained using a Zeiss confocal microscope (Nikon A1 confocal). Image manipulation and merging were performed using appropriate tools of the ImageJ software.