Mooney1 project protocol

Cerebellum and hippocampus acetylcholinesterase activity in males of 20 inbred strains of mice   (2021)

Mooney-Leber S, Gould TJ
With: Zeid D, Garcia-Trevizo P, Seemiller LR, Peltz G

Project protocol - Contents

Workflow and sampling

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)


  1. Mice are euthanized and hippocampus and cerebellum dissected. Hippocampi are further dissected into dorsal and ventral sections (1:1 ratio).
  2. Tissues are flash frozen on dry ice and stored at -80°C until further processing.
  3. 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.
  4. 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)


  1. Total protein concentration of the supernatant from the previous procedure is determined by DC Protein Assay.
  2. Supernatants are further diluted in RIPA buffer to a standard 30 µg total protein.
  3. AChE activity in diluted samples is measured using the Abcam Acetylcholinesterase Activity Kit per manufacturer instructions.