Materials and Reagents

1. Mouse
   Notes:
   1. To reach statistic significance, at least 10 mice are required. 15 mice are ideal to minimize the individual variation.
      
   2. C57BL/6, male, 2 months to one year old (2-4 months old is optimal), 3-5 mice are kept in one cage after weaning.
2. Cleaning solution
   Note: Clean the chamber and/or objects using 70% ethanol first followed by sterile water (to remove the odor of ethanol). Air dry for 3 min.
   

Equipment

1. Apparatus
   Note: The dimensions of plexiglas box is 40 cm x 40 cm x 40 cm. The color of plexiglas is frosted white or transparent. The frosted white plexiglas boxes can diminish the effect from outside views, if the mice have not experienced the open field test.
   
   
   Figure 1. The apparatus for novel object recognition
   
   
2. Objects
   Note: The size of objects is 4 cm x 4 cm x 4 cm approximately. The shape of objects is simple like the building blocks. The objects made by wood, metal or hard plastic are preferred, because those are resistant to biting demolition. Because mice are protanope (red color blindness), the colors of objects should be checked by analysis of Vischeck software (a free software available from the website: http://www.vischeck.com) to ensure that mice are able to distinguish them.
   
3. Camera/computer
   Note: The raw data are stored in recording equipment for further analysis. The video tracking system such as “Smart system” (Panlab) can help you to easily get the exploration time of mice to the objects (the heads of mice crossover the object boundary lines indicated in Figure 1). The exploration time can also be counted manually. The results are further analyzed using the Prism (Graph Pad software).
   

Procedure