Combined immunofluorescence For co-localisation studies, anti-insulin and anti-VP1 immunoreactivity were detected using an AlexaFluor 488-conjugated anti-guinea pig antibody and an AlexaFluor 568-conjugated anti-mouse antibody, respectively (Invitrogen, Paisley, UK). To determine whether the enteroviral VP1 protein co-localised with either PKR or Mcl-1 in beta cells, primary antibodies were incubated as described in ESM Table 3. The primary antibodies were detected with relevant goat secondary antibodies conjugated to AlexaFluor 488 or 568 (Invitrogen) or with goat anti-guinea pig DyLight 405 (Stratech, Newmarket, UK). Control sections were stained with relevant primary and secondary antisera to confirm that no cross-reactivity was detected. Sections were mounted in Vectashield hard-set mounting medium (Vector Laboratories, Peterborough, UK) under glass coverslips. Images were captured using a Nikon Eclipse 80i microscope (Nikon, Kingston upon Thames, UK) and overlaid using NIS-Elements BR 3.0 software (Nikon) to study the relative localisation of each antigen. Sections directly adjacent to those stained using the combined method were stained with an anti-glucagon (rabbit; Dako) or an anti-insulin (guinea pig; Dako) antibody using a standard immunoperoxidase technique to determine total islet numbers in the sections and to distinguish insulin-containing islets (ICIs) from insulin-deficient islets (IDIs).