3D imaging of PSD-95 in the mouse brain using the advanced CUBIC method

Aims Postsynaptic density – 95 kDa protein (PSD95) is an important molecule on the postsynaptic membrane. It interacts with many other proteins and plays a pivotal role in learning and memory formation. Its distribution in the brain has been studied previously using in situ hybridization as well as immunohistochemistry . However, these studies are based on 2 dimensional (2D) sections and results are presented with a few sections. The present study aims to show PSD-95 distribution in 3 dimensions (3D) without slicing the brain tissue of C57BL/6 mice into sections using the advanced CUBIC technique. Methods Immunofluorescent staining using a PSD-95 antibody was performed on a half of the mouse brain after clarifying it using the advanced CUBIC protocol. The brain tissue was imaged using a Zeiss Z1 light sheet microscope and 3D reconstruction was completed using the Arivis Vision 4 dimensional (4D) software. Results The majority of brain nuclei have similar distribution pattern to what has been reported from in situ hybridization and immunohistochemical studies in the mouse. The signal can be easily followed in the 3D and their spatial relationship with adjacent structures clearly demarcated. In the present study, some fiber bundles also showed strong PSD-95 signal, which is different from what was shown in previous studies and need to be confirmed in future studies.