Figure 3. Demonstration of passive CLARITY and immunofluorescent labelling of different neuronal subcompartments, blood vessels and mitochondria.

250 μm human cerebellar sections from control individuals and a patient with mitochondrial disease underwent passive clearing at 37 °C for 4 weeks (a). The quality of immunofluorescent staining is determined by duration of passive clearing; 2 weeks of passive clearing produced minimal labelling of the white matter in the granule cell layer (NF-H; green; 488 nm and MBP; red, 546 nm) with an absence of labelling of mitochondria (COXI; purple; 647 nm; (b)). Extending passive clearing to 4 weeks improved the quality of stain with identifiable Purkinje cells and their axons (NF-H, green; 488 nm) and their myelin sheaths (MBP; red, 546 nm) and mitochondria (COXI; purple; 647 nm; (b)). In cerebellum subjected to passive clearing for four weeks, positive labelling of mitochondria (COXI; purple; 647 nm) and myelinated Purkinje cell axons (NF-H; green; 488 nm and MBP; red; 546 nm) were identified post-mortem human cerebellum tissue from controls and patients with mitochondrial disease (c). Using a combination of antibodies, it is possible to label neurons and track their three dimensional projections, including Purkinje cell (NF-H), axons (NF-H and MBP) and their dendritic arborisations (NF-H; (d)). It is also possible to detect the vascular network (Glut-1 and α-smooth muscle actin; (d)). Scale: 100 μm.