Cf 488a tunel assay apoptosis detection kit

Pancreata were fixed with 4% PFA at room temperature for 1 h, embedded in 30% sucrose and frozen in OCT (Tissue-Tek). Pancreatic sections (10 µm) were stained using a standard protocol. The following primary antibodies and dilutions were used: Guinea Pig anti-Insulin,1:800, (Dako), Rabbit anti-Glucagon, 1:200, (Cell Signaling), Goat anti-Somatostatin, 1:100, (Santa Cruz), Rabbit anti-Urocortin3, 1:500, (Phoenix) and Rabbit anti-MafA, 1:200, (Cell Signaling). The following secondary antibodies were used: Donkey anti-Guinea Pig 594 (Jackson), Donkey anti-Guinea Pig 647 (Jackson), Donkey anti-Rabbit 488 (Invitrogen), Donkey anti- Rabbit 594 (Invitrogen), Donkey anti-goat 647 (Invitrogen). TUNEL labeling was performed using the CF488A TUNEL Assay Apoptosis Detection Kit (Biotium). Slides were imaged using a Leica SP8 Scanning Confocal microscope or a Zeiss Axio Observer.Z1 microscope.

Apoptosis was detected by the TUNEL assay, according to the guidelines of the manufacturer (CF™488A TUNEL Assay Apoptosis Detection Kit, Biotium, Hayward, CA)⁸. Briefly, cells were grown on glass coverslips and, after treatment, they were washed trice with PBS, then methanol-fixed at −20 °C for 15 min. Fixed cells were washed trice with 0.01% (V/V) Triton X-100 in PBS and incubated with 100 µL of TUNEL equilibration buffer for 5 min. After its removal, 50 µL of TUNEL reaction mixture containing 1 µL of terminal deoxynucleotidyl transferase (TdT) were added to each sample and incubated in a dark and humidified chamber for 2 h at 37 °C. Samples were washed trice with ice-cold phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and 5 mg/mL bovine serum albumin (BSA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (0.2 µg/mL) counterstain was performed (10 min, 37 °C, dark and humidified conditions). After three additional washes with cold PBS, one drop of mounting solution was added, then they were observed and imaged under a fluorescence microscope (Leica DM 6000, Leica, Frankfurt am Main, Germany) (20× magnification) with excitation/emission wavelength maxima of 490 nm/515 nm (CF^TM488A) or 350 nm/460 nm (DAPI). Representative fields were shown. The experiments were repeated three times⁴¹.

A TUNEL assay was used for cells apoptosis detection, following the manufacturer’s protocols (CF™488A TUNEL Assay Apoptosis Detection Kit, Biotium, Hayward, CA, USA), with some changes. Briefly, cells were seeded and then processed, as described [47 (link)]. DAPI (0.2 μg/mL, Sigma Aldrich, Milan, Italy) staining was adopted for nuclei. A fluorescence microscope (Leica DM 6000) was used for fluorescence detection (20x magnification). LAS-X software was used to acquire and process all the images that are representative of three different experiments.