2 methylbutane

Mice were euthanized by cervical dislocation, and brains were immediately removed and flash‐frozen in isopentane (2‐methylbutane, Acros Organics) pre‐cooled at −80°C and kept on dry ice. Brains were then stored at −80°C until cryosectioning. Twenty micrometre‐thick serial coronal sections were obtained using a cryostat and thaw‐mounted onto charged slides (Superfrost Plus, Fisher). Slides were left to dry at RT overnight, prior to storage at −20°C. All sections were post‐fixed in formalin (10% buffered formalin phosphate, Fisher) for 5 min at RT prior to use. Volumetric analyses were performed from serial photomicrographs taken with an Olympus BX60 microscope coupled to a Cool Snap Image Pro colour camera, using the Cavalieri principle (Henery & Mayhew, 1989). Details are provided in the Appendix—Materials and Methods.

At the end of the study period, EAE mice were deeply anesthetized with an i.p. injection of ketamine (10 mg ml⁻¹, Boehringer Ingelheim) plus xylazine (1.17 mg ml⁻¹, Bayer) and transcardially perfused with saline (0.9% NaCl + 5 M EDTA) for 5 min followed by 4% PFA for an additional 5 min. The spinal columns were isolated and post-fixed in 4% PFA at 4 °C overnight. The next day, the spinal cords were extracted, washed in 1× PBS and immersed in a 30% sucrose (in PBS) cryoprotectant solution for at least 72 h at 4 °C. The spinal cords were then embedded in optimum cutting temperature medium in a plastic mould and frozen on 2-methylbutane (Thermo Fisher Scientific) on dry ice. The tissue blocks were cryo-sectioned (30 μm for Cx3cr1-YFP^creERT2R26^tdTomato EAE mice, 10 μm for all of the other EAE experiments) using a cryostat (Leica, CM1850) with a microtome blade (Feather) onto Superfrost Plus slides (Thermo Fisher Scientific). The sections were stored at −80 °C until use.
Human tissue was obtained from the Multiple Sclerosis and Parkinson’s Tissue Bank (Imperial College London) under ethical approval (IRAS reference: 279989). Autopsy flash-frozen brain tissue from one case with secondary progressive MS (44 years old, male) was cryo-sectioned with a 10 μm thickness using a cryostat (CM1850, Leica) with a microtome blade (A35, Feather). The sections were stored at −80 °C until use.

Mice were rapidly decapitated, and their brains were flash-frozen in 2-methylbutane (Thermo Fisher Scientific, Hampton, NH, United States) chilled with dry ice. Bilateral punches of the NAcc were excised from 1-mm-thick coronal slices starting from sections corresponding to Plate 14 of the mouse brain atlas (Franklin and Paxinos, 2007 ) and processed for western blot as before (Manitt et al., 2010 (link); Cuesta et al., 2018 (link)). Briefly, protein samples (15 μg) were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane which was incubated overnight at 4°C with antibodies against Netrin-1 (1:7500, Cat#554223, BD Pharmingen, Mississauga, ON, Canada) and β-actin (1:15,000, Sigma-Aldrich, Oakville, ON, Canada). All the samples of the experiment were run and developed in parallel. To calculate the fold change, the optical density (OD) obtained for each band of Netrin-1 was normalized using the corresponding actin OD. To normalize the data to P21, the average of the Netrin-1 OD/actin OD ratio obtained for each animal was calculated and used as a reference to normalize all the ratios obtained in the experiment.

Mice were sacrificed following the completion of behavioral testing. This was conducted in one of two ways: (1) transcardial perfusion with PBS (Life Technologies, Carlsbad, CA, USA) followed by 4% paraformaldehyde (PFA; Sigma-Aldrich), post-fixation in 4% PFA overnight, and then storage in 20% sucrose until embedded in Tissue-Tek OCT compound (VWR, West Chester, PA, USA) and frozen at −80 °C, as previously described [20 (link),39 (link)]; (2) cervical dislocation and decapitation, followed by rapid extraction and flash freezing of the brain tissue in 2-methylbutane (Thermo Fisher Scientific, Asheville, NC, USA) placed in a dry ice and 70% EtOH bath, and storage at −80 °C until use for analysis below. In all cases, the brain and body masses of the mice were recorded.