Pannoramic desk digital slide scanner

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).

Dissected salivary glands were fixed in 4% formalin and embedded in paraffin. Sections of salivary glands were mounted on microscope slides. Tissue 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 tissues 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). The cell nuclei were stained with 1 μg/ml 4’, 6’-diamidino-2-phenylindole (DAPI; Invitrogen) in distilled H₂O for 20 min. After washing, the sections were mounted using Lab Vision^TM PermaFluor^TM (Invitrogen) medium under glass coverslips, then viewed and photographed on a Pannoramic DESK Digital Slide Scanner (3D Histech, Budapest, Hungary).

A total of 8 μm heart sections were fixed overnight in Bouin's solution followed by staining with the Masson's Trichrome Staining Kit (Solarbio, China) as per the instructions of the manufacturer. The images were captured with the Pannoramic DESK Digital Slide Scanner (3DHISTECH, Budapest, Hungary) and infarct area was measured by the ratio between the collagen-positive area and total area through (Image J software, Bethesda, MD, USA). For the Triphenyl tetrazolium chloride (TTC) staining, hearts were isolated and fixed in optimal cutting temperature (OCT) (Sakura Finetek Incorporation, Torrance, CA, USA) for 30 min at −20°C. Then, the heart was sectioned into 7 slices of 2 mm thickness from the base of the heart to the apex. The slides were stained at 37°C for 30 min with 0.5% triphenyltetrazolium chloride (Solarbio, China) in saline and the infarcted area exhibits white.