Neurological Regeneration Assessment by Histological Staining

Neurological regeneration was tested by Nissl staining and Luxol fast blue staining [45 (link)] according to the instruction of manufacture. Rats were anesthetized by 3% pentobarbital sodium (42 mg/kg), injection and intracardially perfused with paraformaldehyde. The brain tissues were isolated and fixed in 4% paraformaldehyde for 24 hours, after which the hippocampus and cerebral cortex of the brain tissue were isolated. The brain tissues were dehydrated with 30% sucrose solution at 4°C for 3–5 days. After that, the brain tissues were preserved in opti-mum cutting temperature compound (OCT) for 6 hours at room temperature. Then the brain tissues were sliced into 8 and 20 μm thick sections at −20°C, respectively. Luxol fast blue (LFB) staining can demonstrated myelin. Briefly, 8 μm coronal brain slices were placed in LFB solution (Solarbio, China) at 60°C for 4 hours, then transferred to Lithium Carbonate for 30 seconds, obtained the image under bright field microscope (Leica, Germany). Nissl staining can demonstrate Nissl bodies. Briefly, 20 μm coronal brain slices were dehydrated in graded alcohol, staining by 0.1% cresyl violet (Solarbio, China), dehydrated in graded alcohol and xylene again, then transfer to Nissl Differentiation Solution (Solarbio, China) for 30 seconds, obtained the image under bright field microscope (Leica, Germany).