Pancreatic Tissue Analysis for Insulitis

Pancreatic tissues were harvested and fixed with 4% PFA overnight at 4 °C and tissues were processed and embedded in paraffin blocks as described previously (80 ). Tissue sections were stained with rabbit anti-insulin antibody (Cell Signaling) and counterstained with DAB peroxidase anti-rabbit IgG (Vector Lab). Images were acquired using a Zeiss slide scanner (Zeiss, Germany), and insulitis scoring was performed on 5 slides, each 30 μm apart, from 9 mice/group as previously described (80 ). Immunofluorescence studies were performed according to the protocol described previously (46 (link)) using the following antibodies: guinea pig anti-insulin (Dako), mouse anti-PD-L1 (Proteintech), and rabbit anti-CXCL10 (Invitrogen). Signals were detected by counter staining with the following antibodies: goat anti-guinea pig (1:400; Invitrogen), goat anti-rabbit (1:200; Invitrogen), and goat anti-mouse (1:200; Invitrogen). All sections were counterstained with DAPI to identify nuclei. Images were obtained with a Zeiss LSM 800 confocal microscopy (Carl Zeiss, Germany), the fluorescence intensities were quantified using Image J, and the corrected fluorescence intensities were presented, as described previously (46 (link)).