From Tissue to Morphometry: A Step-by-Step Golgi-Cox Workflow for Neuronal and Dendritic Spine Analysis in Adult Mice

Neuronal morphology analysis in health and disease is fundamental for understanding brain function and for assessing the effects of experimental interventions. Golgi-Cox staining remains a classical, yet highly relevant technique for visualizing complete neuronal arbors and dendritic spines. However, published protocols frequently differ in critical procedural steps and often lack essential methodological details, leading to substantial variability in staining quality and making laboratory standardization time-consuming and challenging. Here, a comprehensive, step-by-step Golgi-Cox protocol for adult mice is presented, encompassing the entire workflow from in vivo handling and tissue processing to imaging and morphometric data extraction. It details practical considerations and common pitfalls that influence impregnation efficiency, section integrity, and signal-to-noise ratio, and provide methodological guidance to improve reproducibility. Overall, this protocol enables reliable visualization and quantitative analysis of whole neurons, dendritic segments, and dendritic spines. It is anticipated that this resource will facilitate the implementation of Golgi-Cox staining with greater consistency, transparency, and technical rigor, while offering a clear framework for adapting the method to diverse laboratory settings.