Immunofluorescence staining of GFAP and Iba-1

Immunofluorescence staining was performed using 14 µm-thick striatum sections to quantify the number of activated GFAP-positive astrocytes and Iba-1-positive microglia. The brain sections were first washed twice with PBS and incubated with blocking reagent (10 % normal goat serum in PBS containing 0.3 % Triton-X 100) for 1 h. The sections were then incubated overnight with the primary polyclonal rabbit antibodies ant-GFAP (1:1000) and anti-Iba-1 (1:1000) at 4 °C. The sections were washed and incubated with fluorescent secondary anti-rabbit Alexa fluor 488 antibody for 1 h at room temperature. Sections were then washed, mounted on slides, and coverslipped using the mounting medium Fluoroshield (Sigma Aldrich, St. Louis, MO, USA). The images were captured using a fluorescence microscope EVOS FL (Thermo Fisher Scientific, Waltham, MA, USA).