Human Sample QuantificationsFigures 3A and 3B for quantifications of immunostaining on human donor pancreas sections, 7-10 islets were scored for each donor for each marker: non-diabetic (n = 6), autoantibody-positive (n = 6) and autoantibody-positive T1D (n = 6) and data are represented as mean ± SD of the donors (Figures 3A and 3B) or total islets scored in each group (Figure S3D). Each dot represents a donor for the plots in Figure 3. For donor information see Figure S3A.Figure S3C for quantifications of SA-βgal+ beta cells 15-20 islets were scored from two pairs of age-matched non-diabetic and T1D donors. Comparisons were made between the groups using unpaired, two-tailed T-test and considered significant at p < 0.05.Figure S3D the same data from Figure 3 is shown, except broken down showing each islet stained in the donor groups for each marker. Each dot represents an islet.General comments on IHC: Stainings for different markers were performed from sections cut from at least two different regions of pancreas (e.g. pancreas head, body, tail).

Immunofluorescence Staining of Pancreas SectionsImmunohistochemistry of FFPE mouse and human pancreas sections was performed as described (Dhawan et al., 2015Dhawan S. Tschen S.I. Zeng C. Guo T. Hebrok M. Matveyenko A. Bhushan A. DNA methylation directs functional maturation of pancreatic β cells.J. Clin. Invest. 2015; 125: 2851-2860Crossref PubMed Scopus (58) Google Scholar). Briefly, five micron tissue sections were rehydrated with xylene and graded ethanol washes, immersed for 10 minutes in 1% peroxide and subjected to heat-mediated antigen retrieval with sodium citrate pH 6.0 for 7.5 minutes at 100% and 14 minutes at 50% power in a 1250W microwave. Sections were cooled to room temperature with tap water and then permeabilized with TBS containing 0.1% Triton-X-100 for 5 minutes. Sections were blocked with 2% normal donkey serum in protein block buffer (Dako) for 15 minutes and then incubated overnight at 4°C with primary antibodies. Primary antibodies used for immunohistochemistry are listed in the Key Resources Table. FITC or Cy3-conjugated secondary antibodies (Jackson Immunoresearch) were used to detect primary antibodies and sections were counterstained with DAPI (Vectashield). Images were taken on a Zeiss Axioscope2 wide-field fluorescence microscope with AxioVision software, image processing for figure preparation was performed on ImageJ.