A modernised fluorescent silver method for 3D brain histology using an AIE fluorogenic probe

Abstract The study of the complex structural network in the brain requires scalable, robust, and histologically homogeneous chemical staining in three dimensions (3D) and throughout the whole brain. In this work, we report a novel fluorescent brain staining method, named silver-AIE, which utilised an Ag + -specific aggregation-induced emission (AIE)-active fluorogenic probe to visualise the argyrophilic structure in the brain tissue. We demonstrate that the silver-AIE method surpasses the chromogenic detection step in traditional silver staining and eliminates the associated high background, artefacts, and poor reproducibility. Since this method is fluorescence-based, it also allows image acquisition by fluorescence microscopes and at higher axial resolution using optical sectioning. This silver-AIE method is highly compatible with immunofluorescence staining in paraffin sections, potentially allowing for multiplex imaging. It can also be applied in staining a hydrogel-based cleared brain for 3D imaging of large tissue sections. This modernised silver staining revitalises the traditional silver staining, offering new opportunities for brain staining and imaging.