Golgi-Cox Staining of Hippocampal Dendrites

Golgi-Cox staining was performed using the FD Rapid GolgiStain Kit (FD Neuro Technologies, Columbia, MD, USA), as previously described.^{53 (link)} Briefly, animals were anesthetized, and the brains were taken from the skulls and rinsed in double distilled water. The brains were immersed in impregnation solution, and stored at room temperature for 2 weeks in the dark. The impregnation solution was replaced the following day, and the brains were transferred to Solution C. Solution C was replaced the following day and the brains were stored at 4 °C for 3 days in the dark. Brain sections (120 μm thickness) were generated using a Cryotome (Thermo Electron Cooperation) with the chamber temperature set at −22 °C. Each section was mounted on saline-coated microscope slides using Solution C. After the absorption of excess solution, the sections were allowed to dry naturally at room temperature. The dried sections were processed according to the manufacturer's instructions. Briefly, dendrites within the CA1 subregion of the hippocampus were imaged using a × 100 objective using an Olympus BX-51 microscope (Wetzlar, Germany) with a Leica DFC 280 digital camera (Tokyo, Japan). Dendritic spines were detected along CA1 secondary dendrites starting from their point of origin on the primary dendrite and the counting was performed by an experimenter blinded to the group of each sample.