Cell death detection kit

After fixation and embedding, paraffin sections (3 μm) of pancreas were stained with hematoxylin–eosin (H–E). Sections were observed by a fluorescence microscope (Olympus).
Frozen sections (7 μm) of pancreas were measured by TUNEL staining using the Roche Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA). The stained tissues were viewed with a Leica TCS SP8 confocal laser-scanning microscope (Leica, Wetzlar, Germany).

We performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays using a cell death detection kit from Roche Diagnostics. TUNEL assays allow identification of the cells undergoing cell death where the cleavage of double- and single-stranded DNA is labeled by a fluorescent tag (TMR Red) (White et al., 1994 (link); Mccall and Peterson, 2004 (link)). The fluorescently labeled nucleotides are added to 3′ OH ends in a template-independent manner by terminal deoxynucleotidyl transferase (TdT). The fluorescent-tagged fragmented DNA within a dying cell can be detected by fluorescence microscopy. After secondary antibody staining, eye antennal discs were blocked in 10% normal donkey serum in PBS with 0.2% Triton X-100 (PBT) and processed for TUNEL labeling (Singh et al., 2006 (link)). For each genotype, we counted TUNEL-positive nuclei from five sets of eye imaginal discs to determine dying cell population. We used these cell counts for statistical analysis using Microsoft Excel 2013. We calculated the p-values using Student two-tailed t-test, and the error bars represent standard deviation from mean.

Beta cell proliferation was measured by Ki67 (Novocastra Laboratories, Newcastle, UK) immunostaining in paraffin sections. To measure apoptosis in tissue sections, TUNEL staining was performed using the cell death detection kit from Roche (Brussels, Belgium). Ki67 or TUNEL staining were combined with insulin immunostaining [5 (link)].

Dextran sulfate sodium salt (DSS) and L-serine were purchased from Sigma-Aldrich, (Shanghai, China). The animal diet was purchase from Research Diets, Inc. (New Brunswick, NJ, United States). Cell death detection kit was purchased from Roche (Shanghai, China). ELISA quantitative kits for the detection of IgA, IgG, IgM, IL-1β, IL-6, TNF-α, myeloperoxidase (MPO), and eosinophil peroxidase (EPO) concentrations were purchased from Cusabio Biotech (Wuhan, Hubei, China¹). The QIAamp DNA stool MiniKit and Gel Extraction Kit were purchased from Qiagen (Hilden, Germany). The Protein Extract Kit was purchased from Keygen (Nanjing, China). Caspase 3, proliferating cell nuclear antigen (PCNA), mammalian target of rapamycin complex I (mTORC1), and general control non-derepressible 2 (GCN2) antibodies were purchased from Cell Signaling (Beverly, MA, United States). EZ-ECL was purchased from Biological Industries (Cromwell, CT, United States).

DNA fragmentation was detected by using an in‐situ Cell Death Detection Kit (Roche, South San Francisco, CA). After being fixed with 4% paraformaldehyde for 1 hour, cells were incubated with 3% H₂O₂ and 0.1% Triton X‐100 for 10 minutes. Cells were then washed with PBS, and co‐stained with TUNEL inspection fluid and DAPI. Three random microscopic fields per slide were observed under a fluorescence microscope (Olympus Inc, Tokyo, Japan).

TUNEL staining was carried out using the Cell Death Detection Kit (Roche, San Francisco, CA, United States) according to the manufacturer’s protocol. Briefly, the basal temporal lobes were paraffin-embedded and sectioned (5 μm). Subsequently, brain slides were deparaffinized, dehydrated and incubated with the TUNEL reaction mixture for 1 h at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) mounting medium after being washed three times with PBS at room temperature. Finally, the sections were observed by a fluorescence microscope (Leica, Oberkochen, Germany) and TUNEL-positive cells were counted by a researcher who was blind to the experimental groups.