2.10. Nissl staining

The brain slices were routinely deparaffinized and incubated with 0.5% toluidine blue at 55°C for 40 min, and then the stained brain slices were washed with graded ethanol and xylene. Finally, the neutral resin was used to mount the film and observe it under an optical microscope, ²⁶ and calculated the proportion of neurons of positive Nissl staining in the total number of neurons under the microscope field. Similarly, the count was done by an observer blinded to grouping.