Paraffin sections of formalin-fixed human pancreas and isolated human islets were treated with 0.05% pronase for antigen retrieval, blocked with 2% bovine serum albumin (BSA; Sigma)\/phosphate buffered saline (PBS), incubated overnight (4\u00b0C) with 10E4 (anti-HS) mAb (1\/10; US Biological\/Amsbio), washed and stained with AlexaFluor 488-goat anti-mouse IgM (Thermo Fisher). The same sections were washed, incubated with rabbit anti-human glucagon IgG (Abcam) or guinea-pig anti-pig insulin Ig (Dako), washed and stained with Alexafluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher). The specificity of HS staining was checked on serial sections using IgM\u03ba isotype control (BD Biosciences), instead of 10E4 mAb, together with anti-glucagon or anti-insulin antibody. Nuclei were stained with DAPI (0.2 \u03bcg\/ml; Sigma). Sections were photographed using an automated Axio Observer inverted fluorescence microscope (Zeiss). Merged images were prepared using ZEN (version 2.3) software (Zeiss).