Tunel alexa fluor imaging assay

To determine cell apoptosis in the PDO tissues, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) imaging was performed using the TUNEL Alexa Fluor imaging Assay (Invitrogen), following the manufacturer's instructions. Cryofixed graft tissue sections were fixed in 4% PFA for 15 min at room temperature and then permeabilized in 0.25% Triton X-100 in PBS for 20 min at room temperature. The sections were then incubated at 37 °C with a labeled reaction mixture containing 4.0 μL (TdT) and a nucleotide mixture including 1.0 μL (FITC-12-dUTP) for 1 h under dark conditions. After washing three times, they were counterstained with DAPI. Apoptosis was determined by laser confocal microscopy (LEICA SP8, Germany).

TUNEL was implemented in line with the manufacturer’s instructions with the use of TUNEL Alexa Fluor imaging assay (Invitrogen) [24 (link)]. In a word, miR-NC and miR-381-3p were transfected into OGD-induced HT22 cells which were subsequently seeded into 6 cm culture dishes equipped with cover glasses. An immunostaining fixative was taken to immobilize the cells for 30 to 60 minutes, which were rinsed in PBS once later. An immunostaining detergent was administered for 2 minutes’ incubation in an ice bath. Next, 50 μL of TUNEL detection solution was given to the samples for 60 minutes’ incubation in darkness at 37°C. PBS was utilized to rinse the samples three times. After being sealed with the anti-fluorescence quenching sealing solution, the samples were monitored under a fluorescence microscope with 450–500 nm excitation light and 515–565 nm emission light (green fluorescence). With five fields randomly chosen from each sample, the apoptotic rate was calculated as per the formula: apoptosis rate = apoptotic cells/total cells×100%.