Culturing Mouse Cerebellar Slices

Primary cerebellar cultures were prepared from C57BL/6J mice at postnatal day 10 as previously described [55 (link), 56 (link)]. Briefly, cerebella were embedded in 1% agarose and sagittally sectioned in ice-cold DPBS (Sigma‒Aldrich, G8769) supplemented with 5% glucose (Sigma‒Aldrich, G8769) at 300 μm using a Leica VT1000S vibrating microtome. Slices were immediately placed in 24-well plates on individual 0.4 μm 12 mm diameter Millicell-CM cell culture inserts (Millipore, PICM01250) and grown in 350 μL of cell culture medium containing 50% BME (Thermo Fisher, 21010046), 15% heat-inactivated horse serum (Thermo Fisher, 16050114), 25% Hank's solution (Sigma‒Aldrich, H4641), 0.5% glucose, 1% GlutaMAX™ Supplement (Thermo Fisher, 35050061), 1% penicillin‒streptomycin (Corning, 30-002-CI), N2, and 10 ng/mL PDGF-AA (Sigma‒Aldrich, P3076). The slices were incubated at 37 °C in 5% CO₂. Half-volume media changes were made two days after plating. After 4 days, mouse cerebellar slices were treated with 10 µL of Nef or Ctrl EVs once daily for 2 or 4 days. The slices were fixed with 4% PFA for 15 min, delipidated in ice-cold 2.5% acid methanol for 15 min at − 20 °C, and subsequently processed for immunohistochemistry as described below.