Fd rapid golgistain kittm

Golgi-Cox staining was performed according to protocol using the FD Rapid GolgiStain Kit^TM (FD Neurotechnologies). After three weeks of Golgi impregnation, brains were cut coronally (120 µm) using a Leica Vibratome (Leica VT 1200 s) and mounted onto 1% gelatin-coated slides. Slides were then dehydrated through graded ethanol steps, cleared with RotiHistol (Carl Roth), and mounted with DPX mounting medium on coverslips (#1.5). To quantitatively analyze pyramidal neurons in Golgi-stained slides, impregnated pyramidal cells (8–10 neurons per brain, n = 3 littermate brains per genotype) of layer 2/3 in the somatosensory cortex were selected and imaged with a Nikon Eclipse Ti2 using a ×40 magnification. For analysis, single pyramidal neurons were manually reconstructed using Imaris analysis software (version 9.3.1). The average filament area, filament length, and Sholl intersections were analyzed using the same software. Spine counting was performed using Fiji (v1.52n), spines that started from 100 µm distance of the apical dendrite were counted within a 100 µm segment.

The morphology of dendritic spines was analyzed by the FD Rapid Golgi Stain KitTM (FD NeuroTechnologies-Columbia, MD, USA). The PAG tissues were quickly isolated following rats were euthanized, and soaked in the mixture at room temperature in the dark for 2 weeks. The mixture was prepared with solution A and solution B at the 1:1 ratio, and it was refreshed once within 24 h. Then, the PAG tissues were transferred into solution C at room temperature in the dark for 3 days (the solution changed once after the first 24 h). The vibratome (Leica VT 1200S, Japan) was used to cut the PAG tissues into sections (150 μm thickness). Subsequently, in reference to the instructions of the kit, the sections were stained. After being sealed with a neutral resin, the dendritic spines of the PAG were imaged by a Zeiss microscope (Axio Imager A2) (Gibb and Kolb, 1998 (link)). Throughout the analysis, the experimenters were blinded to the experimental groups.