Immunofluorescence analysis of cleaved caspase-3 and in situ fluorescent TUNEL staining

Mouse skin tissue slides were de-paraffin and incubated with antigen unmasking solution supplemented with 20 μg/mL proteinase K for 5 min at RT. The slides were then washed with PBS and incubated with 4% formaldehyde/PBS for 5 min at RT. After washing with PBS, the samples were next incubated with the in situ cell death detection kit reagents (ab66110, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. The slides were then washed with PBS and incubated with a rabbit polyclonal antibody against cleaved caspase-3 overnight at 4℃ and subsequently incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; Sigma). The TUNEL-positive cells and the number of TUNEL-positive cells that expressed or did not express cleaved caspase-3 were detected using fluorescence microscopy (Eclipse E600).