Islet insulin-stained areas were measured using ImageJ supported by the National Institutes of Health (https://imagej.nih.gov/ij/index.html ). The measurement of total and intra-islet HA-positive (HA+) areas was performed using whole-section imaging as described (16 (link), 18 (link)).
Whole-section imaging was performed using a NanoZoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, NJ, USA). Slides were scanned in bright field with a 20× objective and the digital images imported for analysis using HALO image analysis software (Indica Labs, Albuquerque, NM, USA). Islets were identified by their staining for synaptophysin (SYN). All the islets present in the rat pancreas sections were analyzed. The intra-islet HA+ area represents the HA located within islet area bordered by the endocrine side of the peri-islet capillaries. The total islet HA area is the sum of the intra-islet HA+ and the HA+ area measured around the islets at a 5-nm distance from the endocrine side of the peri-islet capillaries (16 (link)). The outer border of this area was delineated using the HALO annotation tool. The stained areas were measured using HALO software. The percent islet HA+ area is calculated as intra-islet HA+ area/islet area × 100.
Whole-section imaging was performed using a NanoZoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, NJ, USA). Slides were scanned in bright field with a 20× objective and the digital images imported for analysis using HALO image analysis software (Indica Labs, Albuquerque, NM, USA). Islets were identified by their staining for synaptophysin (SYN). All the islets present in the rat pancreas sections were analyzed. The intra-islet HA+ area represents the HA located within islet area bordered by the endocrine side of the peri-islet capillaries. The total islet HA area is the sum of the intra-islet HA+ and the HA+ area measured around the islets at a 5-nm distance from the endocrine side of the peri-islet capillaries (16 (link)). The outer border of this area was delineated using the HALO annotation tool. The stained areas were measured using HALO software. The percent islet HA+ area is calculated as intra-islet HA+ area/islet area × 100.
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