Rabbit anti glut2 polyclonal

Pancreata were dissected from K8^+/+ and K8^+/− mice and frozen in O.C.T, sectioned as described above and stained using specific antibodies as described in Alam et al.20 Mouse anti‐K7 (RCK 105; Progen, Heidelberg, Germany), rat anti‐K8 (Troma I; Developmental Studies Hybridoma Bank, NIH, USA), rabbit anti‐K18 (275, kind gift from Professor J.E. Eriksson), goat anti‐insulin (polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti‐insulin (polyclonal; Santa Cruz Biotechnology) and rabbit anti‐GLUT2 (Polyclonal; Millipore, Temecula, CA, USA) antibodies were used to stain the pancreas tissue. Cell nuclei were counterstained with DRAQ5 (Sigma‐Aldrich, Saint Louis, MI, USA). Secondary antibodies used were as follows: donkey anti‐rabbit Alexa 546, donkey anti‐goat Alexa 488, donkey anti‐rat Alexa 488 and goat anti‐mouse Alexa 488 (Molecular Probes, Eugene, OR, USA). The sections were mounted with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Images were acquired under strict comparable conditions between genotypes and treatments using a SP5 confocal microscope (Leica, Wetzlar, Germany).

Isolated and hand‐picked islets from K8^+/+ and K8^−/− mice were homogenized with a 1‐mL syringe (BD, Franklin Lakes, NJ, USA) and 30‐G needle (Henke Sass Wolf, Tuttlingen, Germany), and samples were prepared for SDS‐PAGE and Western blotting as described.20 Primary antibodies used were as follows: rabbit anti‐insulin (Santa Cruz Biotechnologies), rat anti‐K8 (Troma I; Developmental Studies Hybridoma Bank), rabbit anti‐K18 (275, kind gift from Professor J.E. Eriksson), rat anti‐Hsc70 (Stressgen Bioreagents, Ann Arbor, MI, USA), rabbit anti‐GLUT2 (Polyclonal; Millipore) and rabbit anti‐MFN2 (Sigma‐Aldrich, St. Louis, MO, USA). Anti‐rabbit HRP (Promega Biosciences, San Luis Obispo, CA, USA) and anti‐rat HRP (GE Healthcare, Little Chalfont, UK) secondary antibodies were used. The signal on PVDF‐membranes was developed with ECL developing solution (GE Healthcare) and further exposed to X‐ray films (Fuji, Tokyo, Japan). The Western blot films were then analysed with IMAGE J software (NIH) for individual band quantification.