Fluoreview fv10i

Juvenile, adolescent and adult rats fed a standard diet, CE-2, were perfused in the same manner as described above. The VTA-containing sections (−4.9 mm to −5.8 mm from the bregma) were washed in PBS incubated with a blocking solution (0.1% Triton-X, 0.2% BSA, 0.2% NGS) for 30 min. These sections were then incubated with a mouse monoclonal anti-GAD67 antibody (MAB5406, 1:500, Millipore, CA) overnight at 4 °C. Then, the sections were incubated with Daylight 594-labelled anti-mouse IgG (ThermoFisher, IL) for 30 min, mounted with mount medium on glass slides, and covered with a cover glass. The images were acquired using a confocal laser-scanning microscope (Fluoreview FV10i; Olympus, Osaka, Japan). All images were captures in the same light intensity. GAD-positive neurons were counted from the microscope images and the relative intensity of brightness was measured by NIH image software (Image J 1.48 v, National Institute of Health).

Juvenile and adult rats fed a standard diet, CE-2, were perfused in the same manner as described above. VTA containing sections (−4.9 mm to −5.8 mm from the bregma) were washed in PBS incubated with the blocking solution (0.1% triton-X, 2% BSA, 2% NGS) for 30 min. These sections were then incubated with a rabbit anti-ki67 monoclonal antibody (MA5-14520, 1:50, ThermoFisher, IL) overnight at 4 °C. Then, the sections were incubated with Alexa 488-labelled anti-rabbit IgG (ThermoFisher, IL) for 30 min and mounted with DAPI containing mount medium on glass slides and covered with a cover glass. The images were acquired using a confocal laser-scanning microscope (Fluoreview FV10i; Olympus, Osaka, Japan). Ki67 positive neurons were counted under the microscope.

CTB Alexa Fluor 488 (CTB488: Invitrogen, CA) was injected into the DMH or SCN. The injections were performed in the same manner as described above.
Five days after injection, fasting, refeeding and perfusion were performed in the same manner as described above. The brain sections were prepared from − 2.4 mm to − 14.5 mm from the bregma. The sections were then incubated for 60 min with the blocking solution and incubated with rabbit anti-c-Fos antibody (sc-52: 1:1000, Santa Cruz, CA) overnight. Then, the sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (Vector Laboratories, CA), diluted to 1:500. Following incubation, the sections were incubated with Alexa 594-labelled streptavidin, diluted to 1:500, for 30 min. The sections were mounted with mount medium (DAKO, CA). The images were acquired using a fluorescent microscope (Keyence, Osaka, Japan) or a confocal laser-scanning microscope (Fluoreview FV10i; Olympus, Osaka, Japan).
The numbers of c-Fos-IR cells, CTB-labelled cells, and both immunoreactive and CTB-labelled cells per section were counted from the fluorescence images. The cell count per section was averaged for all sections in each investigated nucleus of each animal.