Cy2 or cy3 labeled secondary antibody

Cultured cells were grown on poly-L-lysine-coated (Sigma- Aldrich, St. Louis, MO, United States) glass cover slides in 24-well plates, fixed with 4% paraformaldehyde in PBS for 30 min, washed with Tris–HCl buffer (pH 7.8) containing 8.4 mM sodium phosphate, 3.5 mM potassium phosphate and 120 mM NaCl, and incubated in the same buffer containing 1% bovine serum albumin (BSA) and 0.2% Triton X- 100 for 10 min. Samples were then incubated with the following primary antibodies overnight at room temperature: rabbit anti-GK (1:100, sc 7908; Santa Cruz Biotechnology), rabbit anti-GKRP (1:100, sc-11416; Santa Cruz Biotechnology). Subsequently, cells were incubated with Cy2- or Cy3 -labeled secondary antibodies (Jackson ImmunoResearch Laboratories), counter stained with the DNA stain, TOPRO-3 (1:1000, Invitrogen), and analyzed using confocal laser microscopy Carl Zeiss, LSM700).

In order to analyze the specificity of the adenovirus in vivo, the animals were injected with the adenovirus, and the brains were collected at 48 and 96 h. The rat brains were fixed in 4% PFA by immersion for 48 h. Free-floating frontal hypothalamic slices of 40 μm thickness were obtained by a cryostat and subsequently processed. Tissues were stained with chicken anti-vimentin (1:200; Millipore, Billerica, MA, USA), mouse anti-GFAP (1:200; Millipore), and rabbit anti-NeuN (1:5000; Abcam, Cambridge, MA, USA) antibody diluted in Tris-HCl buffer (pH 7.8) containing 1% bovine serum albumin. After extensive rinsing, the sections were incubated for 2 h at room temperature with Cy2- or Cy3-labeled secondary antibodies (1:200; Jackson ImmunoResearch Laboratories). TOPRO-3 (1:1000; Invitrogen) was used as nuclei staining. The slides were visualized and captured using confocal laser microscopy LSM 700 (Zeiss).

Cultured cells were grown on poly-L-lysine-coated (Sigma-Aldrich, St. Louis, MO, USA) glass cover slides in 24-well plates, fixed with 4% paraformaldehyde in PBS for 30 min, washed with Tris-HCl buffer (pH 7.8), and incubated in the same buffer containing 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 5 min at 22°C. Samples were then incubated with the following primary antibodies overnight at 22°C: rabbit anti-GK (1∶100, sc 7908; Santa Cruz Biotechnology), rabbit anti-GKRP (1∶100, sc-11416; Santa Cruz Biotechnology), mouse anti-vimentin (1∶200, Dako), rabbit anti-GFAP (1∶200, DAKO), mouse anti- MAP2 (1∶50, Chemicon Temecula, CA), mouse anti- neurofilaments (1∶1, Hybridoma Data Bank), goat anti- DARPP32 (1∶50, Santa Cruz Biotechnology), rabbit anti- GLUT2 (1∶100, Alpha Diagnostic International, San Antonio, TX), chicken anti-MCT1 (1∶100, Millipore, Temecula, CA), rabbit anti-MCT4 (1∶20, Millipore) and anti-Kir1.6 (1∶200, Santa Cruz Biotechnology). Cells were next incubated with Cy²- or Cy³-labeled secondary antibodies (Jackson ImmunoResearch Laboratories), counterstained with the DNA stain, TOPRO-3 (1∶1000, Invitrogen), and analyzed using confocal laser microscopy Carl Zeiss, LSM700).