Cryostat sections (8–10 µm thick) of T. melanosporum FBs at different developmental stages were mounted on poly-l-lysine-coated microscope slides and fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature (RT); then they were washed for 20 min with PBS, dehydrated 2 min in absolute ethanol, and stored at −20 °C until use. Before performing TUNEL reaction, cryopreserved sections were incubated in permeabilization solution (0.1% Triton X-100 in 0.1% Na-citrate) for 2 min at 4 °C. After washing with PBS, the sections were incubated in a humid chamber with complete TUNEL reaction mixture, prepared immediately before use according to the manufacturer’s instructions (“In situ cell death detection kit”; Roche Diagnostics, Mannheim, Germany) for 1 h at 37 °C in the dark. Then they were rinsed three times in PBS and mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame CA, USA) containing DAPI. Positive control sections were permeabilized and treated with DNase I 500 U/ml in 50 mM Tris-HCl buffer, pH 7.5, 10 mM MgCl2, 1 mg/ml bovine serum albumin, for 10 min at RT, before the TUNEL reaction. Negative control sections, after permeabilization, were incubated in label solution (without terminal transferase). Observations and photography were performed with a Zeiss Axio Imager.A2 (Jena, Germany) fluorescence microscope (Microscopy Center, University of L’Aquila, Italy) equipped for epi-illumination with appropriate filters.