*2.6. Histological Staining*

#### 2.6.1. Perfusion and Brain Sectioning

A mixture of ketamine–xylazine (100 mg/kgbw and 10 mg /kgbw) was injected intraperitoneally to induce deep anesthesia. Rats were perfused transcardially with ice-cold normal saline solution (0.9%, for 1.5 min), followed by ice-cold fixative solution containing 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) and 0.25% picric acid (Sigma Aldrich, St. Louis, MO, USA) in 0.1 M phosphate buffer (PB, pH = 7.4, Sigma Aldrich, St. Louis, MO, USA) for 20 min. After perfusion, the brains were removed from the skull and postfixed overnight in 4% PFA. Then, 60 μm-thick coronal sections were cut with a vibratome (VT 1000S, Leica, Nussloch, Germany) and washed in 0.1 M PB.
