Chromium 3 potassium sulphate dodecahydrate

All animals were deeply anaesthetised using pentobarbitone sodium (Lethabarb, Virbac, Milperra, NSW, Australia, 100 mg/kg i.p.) and perfused through the heart via the ascending aorta with 20 ml Ca²⁺-free Tyrode's buffer (37°C), followed by 20 ml of a mixture of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and 0.2% picric acid (Sigma) diluted in 0.16 M phosphate buffer (pH 6.9, 37°C) [65] , [66] and 50 ml of the same fixative at 4°C, the latter for approximately 5 min. The brains were dissected out and postfixed in the same fixative for 90 min at 4°C, and finally immersed for 48 h at 4°C in 10% sucrose dissolved in phosphate buffered saline (PBS, pH 7.4) containing 0.01% sodium azide (Sigma) and 0.02% bacitracin (Sigma), before rapid freezing by CO₂. Sections were cut using a cryostat (Leica CM1850, Wetzlar, Germany) at a thickness of 14 microns, and thaw-mounted on slides coated with 0.5% gelatin- (Sigma) and 0.05% chromium(III) potassium sulphate dodecahydrate (Merck, KGaA, Darmstadt, Germany).

Animals were deeply anaesthetized using pentobarbitone sodium (Lethabarb, Virbac, Milperra, NSW, Australia; 100mg/kg i.p.) and transcardially perfused with Phosphate Buffered Saline (PBS) at 37°C, followed by 4% paraformaldehyde (Sigma) and 0.2% picric. The brains were dissected out and placed in the same fixative for 90 min at 4°C, and finally immersed for 48h at 4°C in 30% sucrose dissolved in 0.1 M phosphate buffer Saline (PBS). The brains were snap frozen and then cut in coronal sections using a cryostat (Leica CM1850 (Leica CM1850, Wetzlar, Germany) at a thickness of 14 μm, thaw-mounted on slides coated with 0.5% gelatin (Sigma) and 0.05% chromium(III) potassium sulphate dodecahydrate (Merck, KGaA, Darmstadt, Germany), and stored at -20°C.