Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain

Tissue clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce MOCAT, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. MOCAT with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain, and is able to be used as the evaluation of disease treatment efficiency. MOCAT also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen. Thus, MOCAT represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.