ICR mice (5- to 6-week-old) were sensitized with 20 μL of anti-DNP-IgE/PBS (25 μg/mL) by intradermal injection in the ears with a 0.5-mL insulin syringe (Terumo, Tokyo, Japan). After 24 h, passive cutaneous anaphylaxis (PCA) reactions were induced by intravenous injection of 200 μL of DNP-BSA/PBS (0.5 mg/mL) containing 1% Evans blue. Samples or vehicle (water) were orally administered by feeding needle 1 h before antigen challenge. Ume extract was dissolved in water and TL was suspended in 0.5% methyl cellulose solution (Wako, Osaka, Japan). Mice were sacrificed under anaesthesia 30 min after antigen challenge, and their ears were removed.
For measurement of Evans blue extravasation, mouse ears were punched by an 8-mm biopsy punch (Kai Medical, Gifu, Japan) and punched ears containing extravasated dye were dissolved in 800 μL of 1 N KOH at 37 °C for 24 h. An equal amount of phosphoric acid/acetone (5:13, v/v) was mixed with ears dissolved in KOH and filtered by a 0.45-μm filter. The absorbance of the extract was measured at 620 nm. For histological observation, remaining ears were fixed in 10% formalin and stained with toluidine blue (paraffin-embedded tissue sections, 4-μm thick).
For measurement of Evans blue extravasation, mouse ears were punched by an 8-mm biopsy punch (Kai Medical, Gifu, Japan) and punched ears containing extravasated dye were dissolved in 800 μL of 1 N KOH at 37 °C for 24 h. An equal amount of phosphoric acid/acetone (5:13, v/v) was mixed with ears dissolved in KOH and filtered by a 0.45-μm filter. The absorbance of the extract was measured at 620 nm. For histological observation, remaining ears were fixed in 10% formalin and stained with toluidine blue (paraffin-embedded tissue sections, 4-μm thick).