Dpx mounting medium

The 60 µm sections were mounted on gelatin subbed slides and Nissl stained using 0.25% thionin (according to our standard protocols [Lavenex et al., 2009 (link); Bliss-Moreau et al., 2017 (link)]). Slides were coverslipped using DPX mounting medium (Millipore Sigma, St. Louis, MO, USA). These sections were scanned (TissueScope LE; Huron Digital Pathology; St. Jacobs, ON, Canada) and digital images were used for analyses. Each image was coded to keep evaluators blind to experimental condition. Anatomical boundaries were determined by comparing Nissl-stained tissue to reference atlases (Saleem and Logothetis, 2012 ; Rohlfing et al., 2012 (link)), and all anatomical analyses were performed using StereoInvestigator (MBF Bioscience, Williston, VT, USA).

For the evaluation of the histological study and degree of neuronal cell death, we performed cresyl violet (Nissl) staining [20 (link),41 (link)]. Slides containing 14 µm tissue sections were washed twice for 15 min in 0.01 M PBS. The slides were then stained using 0.1% cresyl violet solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Then, they were dehydrated by 70% ethanol and absolute ethanol. Furthermore, they were treated with xylene to clear tissues and were mounted with DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA). The sample images were examined by using an Olympus AX70 microscope (Olympus, Tokyo, Japan).

The brains were rapidly removed from the skull after decapitation (n = 3–4/group) and fixed in 4% paraformaldehyde (PFA) for 24 h, cryoprotected and dehydrated in graded sucrose solution (10–30% in 0.2 M PBS, pH 7.4). The 25 µm-thick coronal sections at the level of the caudoputamen and the midbrain were mounted on supefrost glass slides, air-dried for 1–2 h at RT and stored at −20 °C until use.
After rehydration in PBS, the sections were treated with 0.3% hydrogen peroxide for 20 min and washed with PBS for 3 × 5 min. Subsequently, sections were blocked with 5% normal donkey serum at room temperature for 1 h, followed by incubation with primary antibodies overnight at 4 °C (Table 1). The slides were then probed with appropriate secondary antibodies (Table 1) for 2 h at room temperature. The signal was visualized using the 3,3′-S-diaminobenzidine-tetrahydrochloride kit (DAB, Abcam, Cambridge, UK) as a chromogen for HRP-conjugated secondary antibodies. After dehydration in graded ethanol (70–100%) and clearance in xylene, the sections were mounted with the DPX-mounting medium (Sigma Aldrich, USA). The sections were examined under a LEITZ DM RB light microscope (Leica Mikroskopie and Systems GmbH, Wetzlar, Germany) equipped with a LEICA DFC320 CCD camera (Leica Microsystems Ltd., Heerbrugg, Switzerland) and analyzed using LEICA DFC Twain Software (Leica, Wetzlar, Germany).