Detecting Apoptosis in Salivary Glands

Salivary glands were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) of salivary glands were mounted on microscope slides. Sections were then deparaffinized, washed in 100% ethanol, and rehydrated. Samples were washed with PBS. After antigen retrieval with 0.1% Triton X-100, the sections were incubated for 1 h with 1:9 TdT mixed with fluorescent-labeled dUTP at 37 °C, following the instructions of the Roche In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany). After washing 2–3 times with PBS, the sections were stained with 1 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen) in distilled water for 20 min [28 (link)]. After washing, the sections were mounted using Lab Vision™ PermaFluor™ (Invitrogen) medium under glass coverslips, then viewed and photographed in a Pannoramic DESK Digital Slide Scanner (3D HISTECH, Budapest, Hungary) [28 (link)]. The TUNEL mean fluorescence intensity was measured using Image-Pro Plus software (IPP, Maryland, USA).