Step Description Equipment Data collected 1 Mice euthanized and tissues harvested Dissection kit 2 Cerebellum and hippocampus (dorsal and ventral) supernatants prepared. Centrifuge 3 Acetylcholinesterase activity determined for each tissue Abcam Acetylcholinesterase Activity Kit Acetylcholinesterase activity for cerebellum and hippocampus (dorsal and ventral)
Procedure 1: Hippocampus and cerebellum harvest and preparation
Equipment, software, and supplies
- Dissection kit
- Centrifuge
- -80°C freezer
Reagents and solutions
- Dry ice
- RIPA Buffer (Sigma Life Sciences, R0278, St. Louis, MO, USA)
- HALT Protease and Phosphatase Inhibitor Cocktail (Fisher Scientific, #78445, Pittsburgh, PA, USA)
Steps
- Mice are euthanized and hippocampus and cerebellum dissected. Hippocampi are further dissected into dorsal and ventral sections (1:1 ratio).
- Tissues are flash frozen on dry ice and stored at -80°C until further processing.
- Tissues from 5-6 mice (randomly chosen from each strain) are homogenized in 1X RIPA buffer solution with HALT Protease and Phosphatase Inhibitor Cocktail at a ratio of 100:1.
- Homogenates are centrifuged at 14,000 g at 4°C for 30 min.
Procedure 2: Acetylcholinesterase activity
Definitions & Abbreviations: AChE: acetylcholinesterase
Reagents and solutions
- DC Protein Assay (Bio-Rad, #500-0112, Hercules, CA, USA)
- RIPA Buffer (Sigma Life Sciences, R0278, St. Louis, MO, USA)
- Acetylcholinesterase Activity Kit (Abcam, ab 138871, Cambridge, MA, USA)
Steps
- Total protein concentration of the supernatant from the previous procedure is determined by DC Protein Assay.
- Supernatants are further diluted in RIPA buffer to a standard 30 µg total protein.
- AChE activity in diluted samples is measured using the Abcam Acetylcholinesterase Activity Kit per manufacturer instructions.