Pancreatic tissues from Bmp6−/− and WT mice were fixed in 10% formalin at room temperature for 24 h and embedded in paraffin. Sections were cut at 5 µm, deparaffinized in xylene and hydrated in distilled water. To identify morphological changes, the sections were stained with hematoxylin and eosin according to standard methods. To determine the iron levels, the sections were placed in Perl's solution (5% potassium ferrocyanide and 5% HCl) for 30 min at room temperature and counterstained with nuclear fast red (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. For the measurement of collagen deposition, the sections were placed in 0.1% Sirius red solution (Fluka; Honeywell International, Inc.) dissolved in aqueous saturated 1.2% picric acid, pH 2.0, for 1 h, washed twice with acidified water (0.5% acetic acid) and passed through 100% ethanol thrice using standard procedures. Quantitative analysis of collagen deposition was performed using ImageJ software (version 1.51r; National Institutes of Health) as previously described (28). The amount of collagen was expressed as a percentage of the total pancreatic surface. The pancreatic islet diameter was measured using ImageJ software. For immunohistochemistry, rabbit anti-insulin (cat. no. ab181547; dilution, 1:64,000; Abcam), mouse anti-glucagon (cat. no. sc-71152; dilution 1:25; Santa Cruz Biotechnology, Inc.), mouse anti- macrophage (Clone Kim2r; cat. no. ABIN284638; dilution, 1:40; Antibodies online) and rabbit anti-CD15 (Clone FuT4/1478r; Novusbio NBP2-53367, dilution 1:10) antibodies were added, and the samples were incubated at 4°C overnight in a humidified chamber. Micro-polymer IHC Detection kit (cat. no. ab236467; Abcam) was used according to manufacturer's instructions with a goat anti-rabbit secondary antibody incubation for 1 h at room temperature. Images were captured using an Olympus Bx51 light microscope (Olympus Corporation) under ×10 and ×20 magnification. A minimum of five unique fields of view were analyzed per sample of pancreatic tissue obtained from four mice per group.

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