Deadend fluorometric tunel system g3250 kit

TUNEL assay was performed on whole flat-mounted retinas [17 (link)]. The DeadEnd Fluorometric TUNEL System G3250 kit was used to detect RGC apoptosis according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, the eye cups isolated from the anesthetized mice were fixed with 4% PFA overnight at 4 °C. The eye cups were digested with 20 μg/mL proteinase K for 10 min, and then incubated with the equilibration buffer for 10 min at room temperature. The samples were incubated with the rTdT incubation buffer for 60 min at 37 °C, and the reaction was terminated by using the SSC solution. The retinas were incubated with DAPI, and then mounted with the GCL being upturned. TUNEL signals were visualized with a confocal laser-scanning microscope through a 20 × objective (FluoView 1000, Olympus, Monolith, Tokyo, Japan). Serial deep scannings were done only in the GCL according to the DAPI staining. All TUNEL-positive signals that merged well with DAPI in each retina were counted.

To detect neuronal apoptosis, the deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay was performed on whole flat-mounted retinas [35 (link)]. The DeadEnd Fluorometric TUNEL System G3250 kit (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions. The whole retina was analyzed, and all TUNEL-positive signals that merged well with DAPI were counted. The fluorescence images were captured using the confocal microscope through a 20× objective (FluoView 1000, Olympus, Japan).