4.5. Detection of Neuronal Degeneration

Fluoro-Jade B (FJB) staining was performed to evaluate the effect of carvacrol on neuronal degeneration after seizure. FJB staining was carried out as defined by Schmued et al. [59,108]. After cryosection, the tissue cut to a thickness of 30 μm was placed on a slide coated with gelatin. Then, the slide was immersed in 100% and 70% alcohol for 3 and 1 min, respectively. The slide was next washed with DW for 1 min and immersed in 0.06% potassium permanganate for 15 min. Then, it was washed for 1 min with DW and immersed in 0.001% Fluoro-Jade B solution (Histo-Chem Inc., Jefferson, AR, USA) for 30 min and next washed with DW, dried in an oven for at least 30 min, and the slide was covered with DPX (Sigma-Aldrich Co., St. Louis, MO, USA). The stained tissue was observed at 450–490 nm using an Axioscope microscope (Carl Zeiss, Munchen Hallbergmoos, Germany). Every 6 th section was selected from bregma to caudal from 2.92 to 4.56 nm. The quantification of FJB-positive neurons was conducted by a blind observer.