Bx60 light microscope

After behavioral training, rats infused with the shRNA plasmids were euthanized with pentobarbital (150 mg/kg) and perfused with 0.9% saline followed by 30% sucrose–formalin solution. Brains were submerged in 30% sucrose/formalin solution before embedding in paraffin. Coronal slices (4 µm) were washed with xylene (15 min) and dehydrated in ethanol washes. Sections were rinsed in 10% PBS (Sigma-Aldrich, P3813) and heated in citrate–EDTA Buffer (pH 6.0) at 95°C for 40 min. After cooling, brain sections were incubated with protein blocking solution (15 min) (BioGenex, HK112-9KE); then incubated overnight at 4°C with primary antibodies against tubulin β III [TU-20] (Abcam, ab7751; 1:50) and GFP (Abcam, ab6556; 1:2000). The tissue was washed with 10% PBS (Sigma-Aldrich, P3813) and incubated for 1 h with the following secondary antibodies from Life Technologies: Alexa Fluor 555 goat anti-mouse (A-21422, 1:100) and Alexa Fluor 488 goat Anti-rabbit (A-11034, 1:50,000). Images were taken using an Olympus BX60 light microscope and analyzed using the NIS Elements Imaging Software (Nikon).

Fixed pancreatic tissues were cut into 5 µm sections for double immunolabeling of glucagon and insulin. Briefly, each section was dewaxed and immunolabeled for α-cells using a polyclonal glucagon antibody (Dako, Carpinteria, CA) and incubated for 30 min at room temperature. A secondary biotinylated anti-rabbit link antibody (Vector Laboratories, Burlingame, CA, USA) was applied at a 1:1000 dilution, and positive immunolabeling was visualized using the peroxidase diaminobenzidine and substrate chromagen system (Dako Corporation, Carpinteria, CA, USA). Thereafter, β-cells were immunolabeled with a monoclonal insulin antibody (1:10000; Sigma Immunochemicals St. Louis, MO, USA) using the alkaline phosphatase method. This was followed by a separate incubation with a rabbit/mouse link, AP Enzyme Enhancer, and substrate working solution (Envision G/2 System/AP, Rabbit/Mouse Kit). The light microscope was interfaced with a computer via Leica Qwin image analysis software (Leica, Wetzlar, Germany). Stained pancreatic sections were viewed with an X20 objective and images were analysed with an Olympus BX60 light microscope comprised of a mounted Nikon DS-Fi1 digital camera.