Obtaining fine neuron morphology and connections data is extraordinarily useful in understanding the brain's functionality. Golgi staining is a widely used method for revealing neuronal morphology. However, Golgi-Cox-stained tissue is difficult to image in three dimensions and lacks cell-type specificity, limiting its use in neuronal circuit studies. Here, we describe an expansion-based method for rapidly clearing Golgi-Cox-stained tissue. The results show that 1 mm thick Golgi-Cox-stained tissue can be cleared within 6 hours with a well preserved Golgi-Cox-stained signal. At the same time, we found for the first time that the cleared Golgi-Cox-stained samples were compatible with three-dimensional (3D) immunostaining and multi-round immunostaining. By combining the Golgi-Cox staining with tissue clearing and immunostaining, Golgi-Cox-stained tissue could be used for large-volume 3D imaging, identification of cell types of Golgi-Cox-stained cells, and reconstruction of the neural circuits at dendritic spines level. More importantly, these methods could also be applied to samples from human brains, providing a tool for analyzing the neuronal circuit of the human brain.
Keywords: 3D visualization; Golgi-Cox stain; immunostain; multi-round immunostain; tissue clearing.