Validation of an easily applicable three-dimensional immunohistochemical imaging method for a mouse brain using conventional confocal microscopy

Histological analysis has been largely confined to two-dimensional analysis of thin tissue sections, hampering detailed understanding of three-dimensional cellular distribution in biological tissues. Tissue optical clearing methods enable three-dimensional histological analysis by rendering tissues transparent and suitable for microscopic detection of the fluorescent signals inside. Despite their great potential in histological research, the tissue clearing methods are not readily accessible to many researchers because of hazardous chemicals, complicated protocols and advanced microscopy. Furthermore, poor antibody penetration represents an additional major obstacle when performing three-dimensional immunohistochemical studies. Here, we have examined tissue optical clearing of a mouse brain slice by a non-hazardous aqueous solution, ScaleA2. We modified the ScaleA2 solution by increasing the concentration of detergent. A simple immersion in the modified ScaleA2 solution alone enabled highly intense and uniform immunolabeling into deep tissues in three-dimensional immunostaining. Conventional confocal microscopy could image three-dimensional immunostaining of vasculature and astrocytes with fine processes to 1 mm imaging depth. Collectively, our technically straightforward clearing method will facilitate the common application of three-dimensional immunohistochemical analysis in many research fields including neuroscience, expanding our understanding of the detailed spatial cellular distribution underlying the physiology and pathology.