Multiplex ImmunofluorescenceMultiplex immunofluorescence was conducted using 4 primary antibodies to detect all neuroendocrine cells: secretogranin 3 (SCG3), beta-cells (insulin), vascular endothelium (CD34, CD31), and SMA following methods previously described27 (Table 2). Control stains showed that islet vessels were uniformly labeled by both CD34 and CD31 (Supplemental Fig. 1). Paraffin sections were dewaxed and rehydrated with TBS and with 0.05% TBST. Nonspecific binding was blocked by incubation in 10% goat serum in TBST for 1 hr at room temperature. Sections were incubated in primary antibodies to SCG3 for 1 hr at room temperature followed by tyramine signal amplification (TSA) with Opal 520 (PerkinElmer; Waltham, MA) conjugate followed by anti-CD34 for 1 hr at room temperature and TSA with Opal 670 conjugate. Sections were reblocked before incubation with anti-insulin antibody overnight at 4C, followed by TBST washes and incubation with goat antiguinea pig Alexa 405 (ThermoFisher Scientific; Waltham, MA) for 1 hr at room temperature. Tissues were reblocked and mouse anti-SMA-Cy3 antibody applied overnight at 4C. Sections were mounted with ProLong Gold Antifade (Life Technologies; Grand Island, NY). Additional sections were stained in 4 control donors and 8 donors with type 1 diabetes for SCG3, CD3, glucagon, and insulin and insulitis determined as previously described.27

Morphometric AnalysesEndocrine and exocrine vessel morphometric analyses were performed using custom Python scripts created for FIJI software (v2.0; https://fiji.sc).28 Total islet area was identified by thresholding the SCG3+ area and applying morphological filters to acquire a smoothed islet boundary to include all SCG3+, CD34+, and SMA+ areas (Fig. 1). Islets were categorized for insulin immunopositivity (INS+/–) using a background-corrected insulin intensity and visual confirmation for every islet. Islet CD34+ area (%), insulin area (%), and SMA+ area (%) were determined via thresholding of the immunostained area divided by total islet area. Particle analysis was used to determine CD34+ islet vessel density (vessels/μm2 islet area) based on previously described methods for individual vessel counts and area (%).29 The plug-in Geodesic Diameters was used to determine vessel diameters via maximum inscribed circles for each vessel, and average vessel diameter was determined for each islet.30 Similar calculations were performed in the same image field for the peri-islet exocrine microvasculature excluding islets and major vessels and ducts.