Nissl staining

Nissl staining is a method of staining nerve tissue with a basic dye, which mainly combines with nucleic acids. After euthanasia, the rats in each group were perfused to obtain tissues, during which squeezing the tissue was avoided to prevent the false appearance of neuron contraction. Frozen tissue sections were washed twice with distilled water for 30 seconds each time. Tissue sections were placed in a 0.5% toluidine blue staining tank, and remained in a 50–60 °C incubator for 30 minutes. Then, the sections were washed twice with distilled water, differentiated by 95% ethanol for 5 seconds, dehydrated with gradient alcohol, made transparent by xylene, and sealed with neutral gum.