Brain Tissue Preparation for Histology

As previously described [67 (link)], the mice were subjected to transcardial perfusion with ice-cold PBS followed by 4% neutrally buffered paraformaldehyde (NBP). After post fixing the brain in 4% NBP for 72 h, it was washed with PBS and then transferred to 20% sucrose solution for a further 48 h. The brain tissue was frozen in optimal cutting temperature (OCT) (Tissue-Teks O.C.T. Compound Medium, Sakura Finetek USA, Inc., Torrance, CA, USA) and sectioned into 14–16 μm sections in the coronal plane with a CM 3050S cryostat (Leica, Wetzlar, Germany). The sections were thaw mounted on Probe-On positively charged slides (Thermo Fisher Scientific Inc., Waltham, MA, USA).