Tyrosine Hydroxylase Immunohistochemistry

Immunohistochemistry was performed as described previously (Seiler et al., 2016 (link)). The brains were cut at 30 μm on a cryostat (Leica CM 1900) and mounted on Superfrost slides (Thermo Scientific). Sections were heated in citrate buffer for 30 min and blocked with 10% horse serum in 0.1% Triton-PBS. Primary and secondary antibodies were incubated in a 0.1% Triton-PBS solution containing 2.5% horse serum. Slides were incubated with the mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000, Millipore) overnight. After PBS washes, sections were incubated for 2 h with the secondary biotinylated anti-mouse IgG antibody (1:200, Vector Laboratories) and the endogenous peroxidase blocked with a solution of 10% methanol and 3% hydrogen peroxide in PBS. Thereafter, the slides were incubated with an avidin-biotin-complex (7 μl/ml; Vectastain ABC-Peroxidase KIT, Vector Labs) for 1 h and specifically bound antibodies were visualized with a metal-enhanced 3,3′-diaminobenzidine substrate kit (Pierce, 34002, Life Technologies). The sections were dehydrated in alcohol, cleared in xylene and mounted in Eukitt (O. Kindler GmbH, Freiburg, Germany).