Basic NeuroscienceConsistent and reproducible staining of glia by a modified Golgi-Cox method

•Visualization of entire glial cytoarchitecture is possible with our modified Golgi-Cox method.•Intensive fixation is crucial to achieve pure glial staining.•Impregnation time (<16 days) and temperature (26 °C ± 1) are critical to attain uniform staining.

BackgroundGolgi-Cox staining is a powerful histochemical approach which has been used extensively to visualize the morphology of neurons and glia. However, its usage as a first-choice method is hindered by its uncertain nature, diminished consistency and lengthy staining duration. The FD Rapid GolgiStain™ Kit (FD Neurotechnologies, Inc., USA) has been developed by employing the Golgi-Cox approach. It is a simple, reliable and reproducible way of performing Golgi impregnation for the analysis of neuronal morphology.New methodWe report here simple modifications to the manufacturer's protocol which enable reproducible and reliable staining of glial cells.ResultsExposure of brain tissue to 4% paraformaldehyde (PFA) during perfusion followed by postfixation with 8% glutaraldehyde in 4% PFA led to only glial cells being stained, whereas in the absence of postfixation both neurons and glia were stained with unclear morphology. Additionally, we found that impregnation at 26 °C ± 1 was critical to attain uniform staining.Comparison with existing methodOur modified Golgi-Cox approach is consistent and reproducible and affords uniform glial staining throughout the brain.ConclusionAs this protocol stains only a small percentage of cells, it is suitable for the analysis of individual cells.