Isolated pancreata were fixed in 10% (vol/vol) formalin overnight at 4°C and embedded in paraffin wax. Sections (5 μm) were cut and fixed onto Superfrost slides. For staining, five sections per mouse, 25 μm apart, were incubated in Histochoice Clearing Agent and then submerged consecutively in 100, 95, and 70% ethanol to remove the wax. Following washes with water, sections were permeabilized by boiling in a citrate-based antigen unmasking solution (Vector Labs, Peterborough, UK), washed with PBS, and then incubated in PBS-Triton X-100 [PBST, 0.1% (vol/vol)] containing 2% (wt/vol) BSA and 2% (vol/vol) goat and donkey serum for 2 h at room temperature. Sections were then incubated in a humidified chamber at 4°C overnight with guinea pig anti-insulin (1:200; Dako, Ely, UK) and mouse anti-glucagon (1:1,000; Sigma, Dorset, UK). After washing three times in PBST [0.25% (vol/vol)] containing 0.25% (wt/vol) BSA, sections were incubated with Alexa fluor 488 and 568-conjugated secondary antibodies (1:1,000; Invitrogen, Paisley, UK) for 2 h at room temperature in the dark. Sections were then mounted using Vectashield antifade mounting medium containing DAPI (Vector Labs). Slices were imaged in the Imperial College facility for imaging by light microscopy (FILM) (http://www3.imperial.ac.uk/imagingfacility), using a Zeiss Axio Observer inverted widefield microscope with LED illumination. Images were captured with a Hamamatsu Flash 4.0 fast camera controlled by Zen software (Zeiss, Cambridge, UK). Image analysis was conducted using ImageJ software (1) and an in-house macro as described under supplementary methods (see online).