Cryotomy of Brain Tissues without Fixation

1. Mouse brains were extracted using lobotomic surgical techniques, and placed at 4° C for 12 h in the solution containing 30% sucrose, 50 mM Tris-acetate, 5 mM EDTA (pH 7.4) and supplemented with cØmplete protease inhibitor cocktail tablets (Roche Applied Sciences, Indianapolis, IN, USA).
2. The brain was then prepared for cryotomy using Leica CM840 cryotome pre-cooled at -25° C. 
3. The cryochuck was placed in the chamber and sufficient Optimal Cutting Temperature medium (OCT)was added to the chuck top until the medium was almost frozen. 
4. The brain was then positioned laterally and more OCT was added until the whole brain was covered by OCT and left to freeze for 20 min. 
5. The chuck was then dipped into liquid nitrogen up until the chuck top and the temperature was equilibrated. 
6. The brain was placed into the cryotome and 10 µm slices were cut and placed onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). 
7. The slides were stored at -20° C. 
8. Prior to any assays, the slides were placed in 0.01 % BSA dissolved in phosphate-buffered saline (PBS) at 4° C overnight in order to prevent non-specific binding of proteins and antibody onto the slides.