Golgi staining kit

Three mouse brains per group were stained using a Golgi-staining kit following the manufacturer’s protocol (Servicebio, Wuhan, China). The brain tissues were submerged in Gorky's staining solution completely in a cool and ventilated place to avoid light for 14 days (the new staining solution was changed after the first 48 h of immersion and then changed once every 3 days for a total of 14 days). The tissues were then transferred to 15% sucrose–PBS (pH = 7.4) and dehydrated in the dark for 1 day at 4 °C. Later, the tissue was transferred to 30% sucrose–PBS (pH = 7.4) and further dehydrated in the dark for 2 days at 4 °C. Next, the tissues were washed with distilled water for 1 min and then immersed in concentrated ammonia water for 45 min. Then, the tissues were washed again with distilled water for 1 min and immersed in an acid film fixer for 45 min. The Golgi-stained brains were sectioned to 100 μm using a frozen microtome (Thermo, CryoStar NX50). The sections were visualized under an upright microscope (Nikon Eclipse E100 microscope). For spine analysis, the dendrites in 20–30 μm lengths of the three tertiary segments were used to measure dendritic spine density via ImageJ (NIH Image J system, Bethesda, MD).

After sacrificing the rats, the brains of 3 rats in each group were removed and placed immediately in 4% paraformaldehyde fixative (Servicebio, G1101) for more than 24 h and subsequently stained by Golgi staining (Golgi staining kit, Servicebio, G1069) and then photographed for analysis using the Image J 6.0 software [National Institute of Health (NIH), Bethesda, MD, United States] (Theer et al., 2014 (link); Louth et al., 2017 (link)). Sholl analysis was used to analyze the dendrite length and branches. The CA1, CA2/3, and DG in HIP and PFC were analyzed. Briefly, the cell body of the neuron was used as the center in the 200× field of view. Then, concentric circles were made separated by a distance of 10 μm, and the sum of the intersection points of the dendrites and concentric circles (data of 10 concentric circles outside the cell) was counted. The total number of intersection points counted was considered to reflect the dendrites and the density of dendritic spines on the base dendrite in the field of 1000× was observed. Due to the uneven distribution of dendritic spines on the dendrites at all levels, observation was started from the first branch of the dendrite to the cell body, and the range of 30–90 μm was used for calculation. The number of internal dendritic spines is the density of dendritic spines per 10 μm.

After the rats (n = 4 per groups) were sacrificed and perfused with normal saline, the brain was removed and immediately fixed in 30% sucrose solution for 48 h. Then, the brain was treated with a Golgi staining kit (g1069, Servicebio, China) for 5 days. After staining, tissue blocks were cut into 50-µm-thick sections, and then sections were dehydrated in absolute ethanol twice for 20 min and cleared in xylene for 30 min. The images were obtained using a VS120-S6-W system (OLYMPUS, Japan) and analyzed with Olympia ver. 2.9.