Materials and Reagents

The protocol was approved by the committee on Animal Health and Care from the University of Amsterdam, the Netherlands (permit number: DED 291).

1. Standard sawdust bedding (just enough to cover the bottom of the box)
   Note: The amount of bedding should be enough to cover the bottom of the context to reduce anxiety behavior. However, this may interfere with other ethological behaviours (e.g., digging), which in turn makes the mouse not to focus on the object but instead with the bedding. You can also try without bedding, but you need to test this before yourself.
   
2. Mouse, C57BL/6J strain, male and female, 2-4 months old, group housed (2-4 mice per group)
   Note: Mice were kept in a temperature and humidity controlled facility (21.5-22 °C, with humidity between 40 and 60%) on a 12 h light/dark cycle (lights on at 8:00 a.m.) with food and water available ad libitum.
   
3. 70% ethanol (v/v)
   Note: Clean the objects using 70% ethanol. Air dry for 1 min.
   

Equipment

1. Apparatus
   For context we used four blue-colored plastic boxes of identical size (W x L x H = 33 x 54 x 37 cm) with or without visual cues on the walls. As visual cues we used tape in white color drawing cues in the walls (Figure 1).
   Note: Consider a large open field apparatus for this test, because the displacement of an object is more noticeable compared to a small size apparatus, while at the same time you provide the mouse with the ability to explore the extra cues in the box.
   
   
   Figure 1. Images for Objects and contexts. A. Day 2: training in context A (Lego objects) for 10 min. B. Day 2: training in context B (bottle objects) - > 10 min. C. Day 3: testing in context B (replaced bottle with Lego from context A) - > 10 min.
   
   
2. Objects
   We used blocks of Lego and/or small bottles (Figure 1).
   Note: The objects should be approximately of equal size to the mouse or slightly bigger. The two set of objects should be different enough to be distinguished by the mice. The objects are recommended to be made of hard plastic or glass, so they are resistant to biting. Both two set of objects should be of approximately equal size.
   

Procedure