Immunofluorescence Staining of Pancreatic Sections

H&E and immunofluorescence staining of frozen pancreatic sections was performed as described previously.8 (link), 18 (link) For immunofluorescence staining, the following antibodies were used: polyclonal guinea pig anti-insulin serum (cat. no. A0564; Dako, Carpinteria, CA, USA), rat anti-CD8 (cat. no. MCA2694; AbD Serotec, Oxford, UK), or rat anti-CD4 (cat. no. MCA1767GA; AbD Serotec), anti-guinea pig IgG-fluorescein isothiocyanate (FITC) (cat. no. F-6261; Sigma-Aldrich, St. Louis, MO, USA) and anti-rat IgG-TRITC (cat. no. T4280; Sigma-Aldrich). Sections were covered with Cytoseal60 mounting medium (cat. no. 18006, Electron Microscopy Sciences, Hatfield, PA, USA). Images were captured with an Olympus IX71 fluorescence microscope equipped with a digital camera (C4742, Hamamatsu). Editing of the pictures was performed using ImageJ software (https://imagej.nih.gov/if/). Images of H&E-stained sections were acquired on a light microscope (Leica, Germany) equipped with a digital camera and Leica Application Suite software (Leica Microsystems, Switzerland).