A Rapid Heat‐Enhanced Golgi‐Cox Staining Method for Detailed Neuroanatomical Analysis Coupled With Immunostaining

ABSTRACT The Golgi‐Cox staining technique is renowned for its ability to delineate neuronal architecture with remarkable precision. However, the traditional protocol's lengthy processing timeline and limited compatibility with immunostaining and transgenic labeling have hindered its widespread adoption in modern neuroscience research. In the current study, we found that adjusting the incubation temperature to 55°C significantly reduced the staining duration to a mere 24 h for 100 µm‐thick sections of mouse brain tissue. Importantly, our optimized protocol is compatible with immunostaining techniques and transgenic mouse models. In addition, using a lipopolysaccharides‐induced mouse model of depression, we found a reduction in dendritic spines labeled by Golgi‐Cox staining and an increase in the number of microglial cells labeled by immunofluorescence in the same samples, in addition, cross‐talk between Golgi‐Cox‐stained neurons and microglial fibers were observed. In conclusion, the modified Golgi‐Cox staining technique allows for the acquisition of a more comprehensive set of data from the same biological tissue with increased efficiency. This advancement promises to improve methodologies in histopathology and neurobiology, making advanced applications of Golgi‐Cox staining more accessible in contemporary neuroscience research.