Golgi-Cox Staining of Hippocampal Dendrites

To analyze the changes in the morphology of the dendritic spines in hippocampus, the Golgi-Cox staining was applied with minor modification as described by Hu et al. [12 (link)]. Briefly, brains stored at 37°C in dark place for two days in Golgi-Cox solution were sectioned at 200 μm in 6% sucrose with a vibratome (VT1000S, Leica, GER). All sections were collected on 2% gelatin-coated slides. Then slices were stained with ammonia for 60 min, washed with water for three times, followed by Kodak Film Fix for 30 min, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. The pyramidal neurons in hippocampal region were imaged with a Nikon microscope (Nikon Eclipse 80i, Japan) using a 40x objective. The spines counted in the present study were on 2-3 stretches of the secondary dendrite about 10 μm in length. About 10–15 neurons from one animal were selected to quantify the spine density. Generally, brains were longitudinally cut into two halves and one hemisphere was processed for morphological staining and the other hemisphere was used to examine special proteins and genes expression.