Insulin

Islet graft and pancreas sections were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound, and frozen on dry ice. Embedded grafts were sectioned on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Standard immunofluorescent staining techniques were used on 6 µm sections. Sections were blocked in 5% BSA for 30 min, incubated with primary antibodies overnight at 4 °C, washed before incubation with secondary antibodies for 2 h at room temperature, and finally washed again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Crus Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher) and color channels were merged using Fiji (http://fiji.sc/). Primary antibodies (1:100): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (Catalog #: a0564) and somatostatin (Catalog #: A0566) from Dako (Carpinteria, CA), glucagon (Catalog #: PA5-88091) from ThermoFisher. Secondary antibodies (1:1000): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO); Alexa Fluor-594 α-Rat, Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.

The chemicals, reagents, and antibodies are the following: phloretin and ruthenium (III) chloride hydrate, DPPH, penicillin, TPTZ, ABTS, CT-DNA (calf thymus DNA), foetal bovine serum (FBS), sodium pyruvate, MTT, Annexin V, propidium iodide (PI) (procured from Sigma-Aldrich Chemical Co.), DMH (dimethylhydrazine), streptomycin, DSS (dextran sodium sulfate), insulin, L-glutamine, streptavidin peroxidase, 3,3′-diaminobenzidine (DAB), and proteinase K. Antibodies specific for p53, PCNA, pro- and active caspase-3, Akt1, phospho Akt, mTOR, phospho mTOR, and VEGF (627501) were acquired from BioLegend (San Diego, CA, USA). Anti-rat antibody for Bcl-2, Bax, beta catenin, NF-κB, and goat anti-rabbit IgG secondary antibody were bought from Genetex, Inc. (Irvine, CA, USA). An immunohistochemistry kit was acquired from Biovision, Inc. (Milpitas, CA, USA), and an apoptosis detection kit was purchased from Takara Bio Inc. (Shiga, Japan).