Fig. 2.

Non-immunohistochemical staining with tissue clearing on formalin-fixed brain tissues. a Colour depth-coded image with maximum intensity projection of a piece of 1-mm-thick medulla block stained with cresyl violet and cleared with OPTIClear (z-stack depth = 228.83 µm). Lower image showing a zoomed and tilted view of area outlined by the white box. b A piece of frontal cortical block immunostained for ZO-1 (green) and counterstained with DyLight 649-labelled Lycopersicon esculentum lectin (red). c DiI crystals were inserted into a cerebellar folium to trace the fibres up to the granular layer, followed by tissue clearing with OPTIClear for 6 h at 37 °C. Most of the fibres traced were mossy fibres. The inset shows the gross appearance of the sample with red crystals of DiI inserted within the OPTICleared sample. d Enlarged view of the white boxed region in a. with colour depth-coding (Z-depth 151.09 μm). e Z-stack images of dendritic spine-like projections visualised in areas outlined by white boxes. Scale bars, 1 μm