Preparation of cashew patches and application to mice.
Epicutaneous patches were loaded with 50 µg of cashew protein extract conjugated to Fluoroprobe-647 (Interchim), prepared in phosphate buffer 0.1 M, or 50 µl of phosphate buffer 0.1 M alone (excipient patches). 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). Patches were maintained for 6 hours based on optimal timepoints previously defined.