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5% agarose gel and visualized by ethidium bromide staining. Nested-PCR products were further harvested and purified using NecleoSpin Extract II (Macherey-nagel) and sequenced by GenScript Corp. Pancreatic immunohistochemistry was conducted according to previous reports [10, 33, 43, 44]. In brief, male C57 mice heart were perfused with physiological saline (0.9% NaCl) followed by 4% paraformaldehyde in phosphate-buffered saline (PBS) containing (in?mm) 137 NaCl, 2.7 KCl, 1.5 KH2PO4, 10.1 Na2HPO4 (pH?=?7.4). The pancreas was then removed and immersed in the same fixative for an additional 6 h. After fixation, the pancreatic tissues were placed in PBS containing 30% sucrose at 4��C until they sank, embedded in tissue-freezing medium (OCT compound), cryosectioned (35 ?m/section) http://www.selleckchem.com/products/MS-275.html and air-dried. Pancreas sections were blocked in 10% BSA/0.4%Triton/PBS solution (blocking serum) for 30 min at RT and immediately exposed to the primary antibodies for immunostaining for overnight at 4��C. The primary antibodies used were rabbit monoclonal insulin antibody (1:400; Cell https://en.wikipedia.org/wiki/Quinapyramine Signaling) and mouse monoclonal GluR2 antibody (1:500; Millipore). The sections were then rinsed in PBS and incubated with fluorochrome-conjugated secondary antibodies (Alex543, 1:1000; Alex488, 1:1000; Invitrogen) for 1 h at room temperature. Immunocytochemistry images were obtained with a confocal microscope (Olympus) at a resolution of 800 �� 800 pixels with the pinhole set at 1 airy unit. All the parameters used in confocal http://www.selleckchem.com/products/GDC-0449.html microscopy were consistent in each experiment. MetaMorph 5.0 software (Universal Imaging) was used to analyze the cell images. Non-fasting male mice (8 weeks) were euthanized by cervical dislocation and immediately the abdominal cavity was opened and collagenase (2 mg/mL dissolved in Hank's buffer; Roche) was injected into the pancreatic duct. The whole pancreas was extracted and digested for 25 min at 37��C and then washed with collagenase-free solution. Islets were individually selected under a microscope to ensure pure islet preparations and maintained (for