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For immunofluorescence staining, 2 different sections per mouse spaced by at least 120 m were co-stained with rabbit anti-insulin (1:500, Thermofisher, bs-0056R) and mouse anti-glucagon (1:500, Abcam, ab10922) primary antibodies overnight at 4C followed with a 1-hour incubation with the secondary antibodies Alexa Fluor 555 donkey anti-rabbit (1:400, Invitrogen, A21428) and Alexa Fluor 488 goat anti-mouse IgG1 (1:500, Invitrogen, 121121) at room temperature