Microm hm 430

A Thy1-GFP line M transgenic mouse (the Jackson laboratory, stock 007788) was used for preparation of brain slices. After being deeply anesthetized with isoflurane (Piramal), a standard transcardial perfusion was performed first with phosphate-buffered saline (PBS; Invitrogen) followed by 4% paraformaldehyde (PFA; Electron Microscopy Sciences). The mouse brain was collected and immersed in 2% PFA and 15% sucrose in PBS solution overnight at 4°C. The immersion solution was then replaced with 30% sucrose in PBS, and the brain was stored at 4°C. After 24 hours, the whole mouse brain was cut to 100-μm-thick slices on a microtome (Thermo Scientific, Microm HM430). Brain slices were immersed in PBS and then placed on microscope glass slides, and allowed to dry for 45 min. Cover glass (Fisherbrand, No. 1.5, 0.16 to 0.19 mm thick) with mounting medium (Vectashield Hardset Antifade mounting medium, H-1400) was then placed on top of the glass slides with brain slices. Slices were ready for imaging after the mounting medium completely hardened. During imaging, brain slice samples were placed on a 2D goniometer platform (Thorlabs, GNL 10).

Brains were removed, post‐fixed in 4% paraformaldehyde overnight, and stored at 4 °C in a 30% sucrose solution. A sliding microtome (Microm HM 430, Thermo Fisher Scientific, Waltham, MA) was used to section the brains at 20 µm thick in coronal orientation through the dorsal hippocampus. Brain sections were then cryoprotected in an antifreeze solution (30% glycerol + 30 % ethylene glycol + 40 % 0.01 m PBS) for storage at –20 °C. Cresyl‐violet (0.1%, Sigma‐Aldrich) was dissolved in distilled water and filtered. Every third brain section was mounted on poly‐d‐lysine‐coated slides and stained for 20 min with a Cresyl‐violet solution. Sections were then dehydrated for 2 min sequentially with 100, 95, 70, then 50% ethanol, cleared in xylene for another 2 min, covered with DPX mounting media (Sigma‐Aldrich, CA) and coverslipped. Lesion area was assessed on 8 to 12 brain sections spaced equidistance (every 450 mm) apart, approximately between ‐1.70 to ‐2.70 mm from Bregma. Lesion volume was obtained by multiplying the sum of the lesion areas by the distance between sections. Percent of lesion volume was calculated by dividing each lesion volume by the total ipsilateral hemisphere volume (similarly obtained by multiplying the sum of the areas of the ipsilateral hemispheres by the distance between sections).