Immunofluorescence for detection of cleaved caspase-3

The sections were immersed in 0.001 M sodium citrate buffer pH 6.0 and placed into a microwave oven at 94 °C for the antigen retrieval. After a cooling-off period, the slides were washed in 0.01 M PBS pH 7.2 and incubated overnight with polyclonal rabbit anti-caspase-3 antibody (1:200; Abcam, Abcam, Cambridge, UK; ab 13847) in humidified chamber at 4 °C. After washing in PBS, the sections were incubated with Alexa Fluor® 594 goat anti-rabbit antibody (1:1000; Molecular Probes® by Life Technologies, Calrsbad, USA) for 20 min at room temperature. The nuclear staining was performed with DAPI (Molecular Probes by Life Technologies; Carlsbad, California, USA) for 5 min in the dark at room temperature. After washing in PBS, the slides were mounted with Flourmount G medium (EMS, Hatfield, USA). As negative control, the sections were incubated with non-immune serum in place of primary antibody. The sections were examined with a fluorescent microscope DM400 B LED (Leica, Wetzlar, Germany).

The number of cells exhibiting cytoplasmic caspase-3 immunofluorescence was computed in a standardized area (0.073 mm²) from each section. The number of caspase-3-immunolabelled cells per mm² was calculated in all animals from the different groups at 7 and 60 days (n = 5 per group/period).