*4.2. Animals and Diets*

*m-Traf3ip2* and BALB/c mice were previously generated as described in prior studies [5] and purchased from Japan SLC, Inc. (Shizuoka, Japan). SPF-grade animals were maintained under conventional conditions in animal facilities certified by the Animal Care and Use Committee of the Maebashi Institute of Technology. The experimental procedures were all in accordance with the National Institutes of Health guidelines for the care of experimental animals, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Maebashi Institute of Technology (16-001). The animals were housed individually in plastic cages, allowing for the separation and collection of feces, at 21 ◦C with a relative humidity of 55% under a 12:12 h light:dark cycle. 6-week-old mice were housed as 5 mice per cage in a controlled environment. After 1 week of acclimatization, the mice were randomly divided into two groups, and thereafter, were fed the experimental/normal or fucoidan diets with free access to drinking water. The animals were fed Rodent Diet AIN-93G (CLEA Japan, Tokyo, Japan). Two groups of 14 or 9 *Traf3ip2* mutant mice were fed a diet containing 1.0% w/w fucoidan or crystalline cellulose, respectively. Each group was fed for 9 weeks. Two groups of five wild type, BALB/c mice, were fed a diet containing 1.0% w/w fucoidan or crystalline cellulose, respectively. Each group was fed for 31days.

#### *4.3. Analysis of Phenotype and Scratch Test*

The face of each mouse was photographed from three angles: front, left side, and right side. To score the severity of skin inflammation, the severity of the psoriasis-like skin condition in the facial area of *m-Traf3ip2* mice was monitored and graded every 7 days. The scoring system was based on the clinical PASI [38]. The modified PASI score was based on three parameters, namely, erythema, scaling, and thickening. Each parameter score was assigned independently on a scale of 0 to 4 as follows: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked. A scoring table with red taints was used to score the level of erythema. The cumulative score (erythema, desquamation, swelling, and scaling area) denoted the severity of inflammation (scale 0–100%). The modified PASI test was applied to each mouse by five persons. Accurate scratching behavior of individual mice was assessed by counting the scratches. Scratching behavior was monitored and the number of scratching movements was counted during a 10-minute period each week.

#### *4.4. Histochemical Analysis of Facial Skin Dissection*

After shaving the faces of mice, their facial skin was fixed with 4% paraformaldehyde and then OTC-embedded punch biopsies were sectioned longitudinally into 7–8 μm thick sections. The sections were stained using HE for histological evaluation and for the evaluation of the microarchitecture of thickened epithelium and the keratinocyte layer. Experiments were conducted in triplicate; the data were averaged to evaluate inflammation and epithelial and keratinocyte changes.
