According to the manufacturer's instructions, 30 mg of brain tissue was placed in collection tube and homogenized completely in 700 μl Buffer TR then was centrifuged at 14000×g for 3 min.
The fixed brain tissue was placed in a coronal rat brain matrix (Activational Systems Inc., Warren, MI, USA) and cut into 2-mm blocks.
Brain tissue was placed in a glass homogenizer in 0.8 ml of ice cold depletion buffer (10 mM HEPES in HBSS, pH 7.4).
Briefly, brain tissue was placed inside a sealed polycarbonate tube along with PBS and a small volume of 1.0 mm silica beads.
In the study of CO histochemistry plus Nissl's counterstaining, the brain tissues were placed in sucrose/PBS with concentrations of 10 %, 20 and 30% and 30%ially until they sequentially
Brain tissue sections were placed on high tissue-binding glass slides (Superfrost Plus; Thermo Fisher, Waltham, MA), heat-fixed at 55 °C using a slide warmer Premiere XH-2001 (C&A Scientific, Manassas, VA), and subsequently stored at −80 °C.
Brain tissue was then placed in autoclaved eppendorf tubes and kept on dry ice for immediate RNA extraction.
After death confirmation by lack of pulse, breathing, corneal reflex and response to firm toe pinch, inability to hear respiratory sounds and heartbeat by use of a stethoscope then we performed decapitation followed by harvesting of brain tissues which were placed in 10% formaldhyde for 2 hours.
Spleen, thymus and brain tissue was collected and placed in ice-cold PBS/2%FBS.
The four brain tissues were weighed, and placed separately in 5 ml of ice-cold homogenizing solution (8.8 mg of ascorbic acid and 122 mg of EDTA in 1000 ml of perchloric acid 0.1 M).
