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Measurements of energy expenditure and locomotor activity HFD-induced obese C57BL/6J male mice were placed in individual small acryl calorimeter chambers with free access to HFD and water, and respiratory gas (O 2 and CO 2 ) were measured every 5 min for each individual mouse by indirect calorimetry system connected to mass spectrometer (Arco2000, ArcoSystem, Chiba, Japan)

Gently spread an appropriate amount evenly over the skin with your hands or with a cotton pad, avoiding the eye and mouth areas

Avoid touching stoppers with hands or unsterile tools

But even then, the injection site makes a difference