Fjc staining

FJC staining (Merckmillipore, Germany) was performed according to the operation instructions and to detect degenerating neurons. Briefly, frozen sections were prepared, fixed, and immersed in a basic alcohol solution consisting of 1% sodium hydroxide in 80% ethanol for 5 min, then rinsed for 2 min each in 70% ethanol and distilled water, and then incubated in 0.06% potassium permanganate solution for 10 min. Following a 1–2 min water rinse, the slices were transferred for 10 min to a 0.0001% solution of FJC dissolved in 0.1% acetic acid vehicle and then rinsed through three changes of distilled water for 1 min per change. The slices were air-dried, coverslips were applied, and the sections were visualized on an Image J software (Image J 1.4, NIH, USA). Two observers blinded to the experimental group counted the FJC-positive cells in six sections per brain (at 40× magnification) through the injury’s epicenter. The data were presented by the average number of FJC-positive neurons in the fields.

To visualize degenerative neurons, brain sections were subjected to FJC staining according to kit instructions (Millipore, USA). Briefly, the slides were fixed with 4 % paraformaldehyde for 10 min then immersed in 1 % NaOH in 80 % ethanol for 5 min. After rinsing for 2 min each in 70 % ethanol and ddH₂O, slides were incubated in 0.06 % potassium permanganate solution for 10 min, rinsed in ddH₂O, then transferred to the staining solution for 10 min. Post washing, slides were air-dried at 50 °C for 30 min then cleared in xylene and mounted with Entellan New (Sigma-Aldrich, USA).