(e) Tunel assay for detection of apoptotic cell nuclei

Apoptosis was evaluated in the same biological samples previously analysed for BrdU labelling, using serial sections at similar depths in the tissue. Control samples not exposed to BrdU were also assessed for apoptosis (one control per life stage). Apoptosis is characterized by formation of DNA breaks (nick), and the TUNEL reaction uses the enzyme terminal deoxynucleotidyl transferase (TdT) to fill these gaps with fluorescein (FITC)-labelled dUTP. The TUNEL assay was performed for 1 h at 37°C according to kit instructions (in situ cell death detection—fluorescein, Roche). For negative controls, TdT enzyme was omitted from the reactive solution. For positive controls, sections were pre-incubated with recombinant DNAse I (3 U ml⁻¹, Ambion) for 10 min at room temperature. Treated sections were mounted with Prolong Gold Antifade reagent with DAPI (Invitrogen) and observed on a wide-field fluorescence microscope (Olympus XM10 slide scanner, PT-BIOP, EPFL), using DAPI (Ex 377/50 nm, Em 440/40 nm) and FITC filters (Ex 485/20 nm, Em 525/30 nm), at spatial lateral resolution of approximately 0.3 µm. The proportion of TUNEL-positive cell nuclei was calculated from acquired images, using the counter cell plugin from ImageJ software. For each life stage, three biological replicates were assessed, with one to four sections imaged per replicate, and totals of 657 to 8219 nuclei counted per tissue type (depending on section orientation).