Hm505e

Mice were deeply anesthetized with pentobarbital and perfused through the heart with 15–20 ml of phosphate-buffered saline (PBS, pH 7.4) to remove the blood, and immediately followed by 50 ml of ice-cold fixative containing 4% paraformaldehyde and 0.2% glutaraldehyde in PBS. The animals were kept on crushed ice throughout the procedure. After perfusion, the whole brain was removed from the skull and postfixed with the same fixative solution at 4 °C for 2 h, then placed in 5% buffered sucrose at 4 °C overnight. Each brain block was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Japan Co., Ltd) and frozen on liquid nitrogen and stored at −80 °C until use. Frozen tissue blocks were sectioned serially at a thickness of 20 µm in a cryostat HM505E (Carl Zeiss, Germany) at −20 °C, and sliced sections were mounted on 0.5% gelatin-coated glass slides. Brain slices located approximately 0.74 mm rostral from bregma and 4.60 mm caudal from bregma were used for tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2) immunohistochemistry, respectively.