Golgi-Cox Staining for Dendritic Spine Staining and Density Measurement

Mice were perfused transcardially with 0.9% saline followed by 4% PFA. After that, the whole brain was collected and fixed in 4% PFA overnight at room temperature. Next, the brains were transferred to Golgi-Cox solution and performed the staining process as previously described (Gibb and Kolb, 1998) for 5 days at 32°C. Then, the brains were transferred to 30% sucrose solution. The brains were sectioned at a thickness of 60 μm using a vibratome, and 5 sections/brain were collected for examination. Each group consisted of two animals, for each brain, 5 sections were chosen and 6 neurons from each section (3 from left, 3 from the right hippocampus—including DG and CA1) were selected for spine analysis. Neurons with properly stained dendrites were selected and the second to sixth proximal branch of apical dendrites were taken for spine counting. Spines were counted manually using ImageJ (downloaded from National Institutes of Health, Bethesda, MD, USA, website: https://imagej.nih.gov/ij/download.html). Spine density was represented as the number of spines per 10 μm of dendrite for 30–60 dendritic segments per group.