Immunohistochemical Analysis of Phospho-p44/42 MAPK

Immunohistochemistry was performed as described in Papale et al. (2016 (link)). After quenching with 3% H₂O₂, 10% methanol for 15 min, the sections were rinsed in TBS and incubated for 1 h in blocking solution (5% normal goat serum, 0.1% Triton X-100). The sections were then incubated overnight at 4°C with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204; 1:1,000, Cell Signaling Technology, Danvers, MA, USA). On the following day, a biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs) was applied to the sections for 2 h at room temperature. Detection of the bound antibodies was carried out using a standard peroxidase-based method (ABC-kit, Vectastain, Vector Labs), followed by incubation with DAB and H₂O₂ solution. The sections were subsequently dehydrated using increasing concentrations of ethanol and mounted with DPX. Images were acquired from the striatum using a bright-field microscope (Leica DMI6000B Macro/Microimaging system) under a 20× magnification.