Immunohistochemical staining for tyrosine hydroxylase

Perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) was carried out after 5 weeks of surgery. The removed brains were post-fixed in the same fixative at 4 °C overnight and then cryoprotected with 30% sucrose-containing 0.1 M PB at 4 °C followed by frozen in dry-ice. The frozen brains were sectioned into 30-µm-thick. The sections were stocked in 0.01 M phosphate buffered saline (PBS) at 4 °C until use.

Immunofluorescence staining using the free-floating sections were performed as previously described⁵³. The sections were rinsed in 0.01 M PBS and then immersed into 0.01 M PBS containing 0.3% Triton-X and 3% bovine serum albumin at 22 ± 2 °C for 1 h because of increase of permeability to antibody and blocking of non-specific reaction followed by treatment with rabbit anti-Tyrosine hydroxylase polyclonal antibody (1:1000; Catalog no. ab112; Abcam Cambridge, UK) at 4 °C overnight. The sections were washed for several times and then incubated at 22 ± 2 °C for 1 h with donkey anti-rabbit immunoglobulin G antibody conjugated Alexa Fluore 488 (1:500; Catalog no. A-21206; Thermo Fisher Scientific). After washing thoroughly, the samples were mounted on the microscope slides with PermaFluor (Thermo Fisher Scientific). The analysis of stained slides was performed with an Olympus microscope (BX53; Olympus Corporation, Tokyo, Japan) and a Keyence microscope (BZ-X700; Keyence).