Tissue Preparation and Immunostaining

At the time of sacrifice, the mice were perfused with saline containing 2.5 U/ml heparin and fixed with 4 % paraformaldehyde under deep anesthesia. The brains and spinal cords were decalcified with 10 % EDTA (pH 7.2) for 2 weeks and dehydrated in a graded sucrose series. The tissues were embedded in OCT compound (Tissue-Tek®; Sakura, Tokyo, Japan) and stored at −80 °C. Ten micron-thick frozen blocks were sagittally sectioned using a cryostat. The sections were stained with hematoxylin and eosin.
Sectioned tissues were immunostained with anti-human nuclei (1:250; Millipore, Billerica, MA) and/or anti-Ki67 (1:400; Abcam, Cambridge, UK) antibodies as previously described [4 (link)].