Golgi-Cox staining is based on the principle of heavy metallic impregnation of neurons, which allows the visualization of the fine structure of a neuron.1 Dendritic spines are tiny protrusions of neurons first described by Santiago Ramón y Cajal using Golgi staining.9,10 Changes in the morphology and density of dendritic spines are usually regarded as a sign of the dynamics of synaptic function.10,11 We demonstrate the imaging of dendritic spines of the Golgi-Cox-stained Spurr-embedded pyramidal neurons in a mouse hippocampus by using confocal fluorescence microscopy. A brain slice of a seven-week-old male Kunming (KM) mouse was prepared using the modified Golgi-Cox method,12 in which the darkening solution is LiOH instead of ammonium hydroxide. The mouse was deeply anesthetized, and the brain was carefully removed and then placed in a Golgi-Cox solution in the dark at room temperature for fixation and impregnation. The brain was used for more than three-month impregnation and then cut into -thick slices in a microtome cryostat. After 1% LiOH alkali treatment for 30 s, the brain slices were gradually dehydrated, embedded with Spurr (SPI, USA), sealed with cover glass, and then kept in an oven at 60°C for 36 h. After the polymerized brain was cooled to room temperature, it was kept dry in the dark until data acquisition. Neuron morphology in the subcortex was imaged using a 488-nm laser (5-mW output power) and objective (dry, N.A. 0.8); the laser power at the specimen will be lowered to 20 to 30% of the output power. Images were acquired using inverted confocal fluorescence microscopy (LSM710, Zeiss). With Spurr fluorescence illumination, we can observe clear soma and dendrite structures in the entire scanning area [Fig. 2(a)]. An apical dendrite and its spines were imaged using a 488-nm laser (15-mW output power) and a objective (water immersion, N.A. 1.20), and are shown at magnification in Fig. 2(b) [corresponding to the red box in Fig. 2(a)]. The output power of the 488-nm laser in the confocal microscopy is 5 mW when imaging with a objective and 15 mW when imaging with a objective. Depending on the morphology, spines can be classified as follows: stubby (short without neck), thin (thin with a small head and a long neck), mushroom (bulbous head with a narrow neck), and cup-shaped or branching (one neck protruding from dendritic shaft and splitting into two subnecks, and one small head for each subneck).10 In order to show the clear spine morphology of the apical dendrite, in Figs. 2(c) and 2(d), we acquired images of the part shown in the boxes in Fig. 2(b) at magnification by using a water objective with color the inverted using Photoshop software. We can clearly observe the stubby (arrow head), thin (arrow), mushroom (star), and branched (diamond) spines in Figs. 2(c) and 2(d). The results of confocal fluorescence imaging of the stained neurons show that this method is suitable for tracing fine spine structures of pyramidal neurons in the hippocampus. Through the comparison of the fluorescence imaging approach with a conventional Golgi-staining imaging method, the image from the fluorescence imaging approach demonstrates a better, at least comparative, ability to reveal the fine structure of Golgi-stained neurons.