Preparation of cashew patches and application to mice.
Epicutaneous patches were loaded with 50 µg of cashew protein extract (Stallergenes Greer, Lenoir, NC) or 50 µg of cashew protein extract conjugated to Fluoroprobe-647 (Interchim), prepared in phosphate buffer 0.1 M. Patches were dried at 30°C in a ventilated oven and stored at 4°C. Before patch application, mice were anaesthetized with ketamine and xylazine (50 and 10 mg/kg, respectively) and hair on the back was removed using electric clippers and depilatory cream (Reckitt Benckiser). Patches were applied the following day and secured using an Urgoderm® bandage (Urgo Laboratories) for 48 hours. As a negative control, mice received patches loaded with phosphate buffer 0.1 M.