Skin permeability assay.

The skin permeability was evaluated using toluidine blue exclusion assay. In brief, 2-month-old iSkiv2l^–/– mice and Skiv2l^fl/fl littermates were shaved after euthanization and rinsed in PBS. The whole body was dehydrated by immersing in 25%, 50%, and 75% methanol/PBS followed by 100% methanol (1 minute each). The mouse skin was then rehydrated with the same series of methanol/PBS solutions (1 minute each), washed in PBS, and stained in 0.0125% toluidine blue O/PBS for 1 minute. The mice were immediately photographed after a brief wash in PBS. Transepidermal water loss (TEWL) of iSkiv2l^–/– mice and Skiv2l^fl/fl littermates was measured using Vapometer (Delfin Technologies).