Cryotomy of Brain Tissues without Fixation
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- 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).
- The brain was then prepared for cryotomy using Leica CM840 cryotome pre-cooled at -25° C.
- 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.
- 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.
- The chuck was then dipped into liquid nitrogen up until the chuck top and the temperature was equilibrated.
- The brain was placed into the cryotome and 10 µm slices were cut and placed onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany).
- The slides were stored at -20° C.
- 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.
