4.5. IHC Image Capture and Analyses

Images were acquired by fluorescence microscopy (Axioplan2 Imaging; Carl Zeiss Microimaging Inc.). For IHC analyses, sections were anatomically matched with the mouse brain using atlas50 (hypothalamic region: between 1.46 and −1.82 mm from bregma). Both sides of the bilateral hypothalamic region were analyzed for two brain sections per mouse. The number of GFAP-positive astrocytes was counted using ImageJ 1.47 v software (National Institutes of Health, Bethesda, MD, USA; http://rsbweb.nih.gov/ij/). Hoechst (Sigma-Aldrich, St. Louis, MO, USA) staining was performed to identify cell nuclei. In sections double-labeled with GFAP antibody and tomato lectin, the number of astrocytes in contact with blood vessels was calculated as a percentage of the GFAP-positive cells in contact with blood vessels per total number of GFAP-positive cells.