Golgi-Cox staining protocol

Mice were deeply anesthetized with isoflurane and perfused intracardially with phosphate buffered saline (PBS) (15 ml) followed by 4% paraformaldehyde (pH 7.4) (25 ml). Brain and spinal cord were removed and stained with Rapid Golgi according to kit protocol (FD Rapid Golgi-Stain Kit, FD Neuro-Technologies, INC), described as follows. Brain was placed in 5 ml of Solution A and B of ratio 1:1 for 24 hours and then immersed in an identical volume of fresh Solution A and B for an additional 14 days in dark. The samples were then transferred to solution C for 3 days, with a replacement of fresh solution after 24 hours. To attain cryoprotection, brains were then transferred in a 30% sucrose solution in PBS for 1-2 days. Afterward, samples were snap frozen in optimal cutting temperature compound (SakuraTek, Japan) on dry ice and kept frozen at −80°C to be cut into 100 μm thick coronal slices using Cryo-cut 1800 (Reichert Jung) at −14°C. Cut slices were stored in 24-well plates with 0.02% azide in PBS at 4°C. To develop GC staining, brain slices were washed in distilled water (2 x 4 minutes) and submerged in solution D and E according to kit protocol. Slices were rinsed in distilled water (2 x 4 minutes) again.