*4.4. Sample Preparation*

Three and seven days after global cerebral ischemia, all experimental rats were deeply anesthetized with urethane (1.5 g/kg) dissolved in 0.9% normal saline to obtain brain samples. For removal of whole blood, deeply anesthetized experimental animals were intracardially perfused with 0.9% normal saline followed by 4% paraformaldehyde (PFA) dissolved in phosphate-buffered saline (PBS) for brain sample fixation. Terminated sample fixation occurred during perfusion, and the whole brain sample was obtained and was immersed in 4% PFA for one hour. After post-fixation, samples were moved into 30% sucrose for cryoprotection. Brain samples sank to the bottom of the tube, and the whole brain was coronally sectioned into thicknesses of 30 μm each using a cryostat microtome (CM1850, Leica, Wetzlar, Germany).
