Vascular Labeling and 3D Imaging of Spinal Cord

Mice (EAE n = 13, control n = 9) were anesthetized with a ketamine-xylazine mix (Ketamine, 100 mg/kg; Xylazine, 20 mg/kg). Shortly before perfusion-fixation, 50 µl of 1 mg/ml Lycopersicon Esculentum lectin conjugated to DyLight649 (DL-1178, VectorLabs) was transcardially injected [5 (link)]. After 5 min of circulation animals were transcardially perfusion-fixed with phosphate buffered saline (PBS) containing 5 IU/ml heparin followed by 4% Paraformaldehyde (PFA, in 0.2 M phosphate buffer, pH 7.4) for fixation of the tissue. Spinal columns were harvested and post-fixed overnight in 4% PFA. The iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) protocol was carried out as described by Renier et al. (2016). Briefly, the spinal columns were first decalcified in EDTA (10% w/v, pH 8–9, for 24 h). Next, the tissue was dehydrated in increasing methanol/H₂O series (20%, 40%, 60%, 80%, 100%, 100%, 1 h each), delipidated with methanol/dichloromethane (33%/66% for 3 h) and pure dichloromethane (2 × 15 min), and optically cleared with dibenzyl ether (DBE, 100%) for at least 14 days. Lastly DBE was replaced with ethyl cinnamate (ECi, 100%) at least 7 days prior to imaging.