*2.2. Immunohistochemistry*

Serial 3-μm para ffin sections were used for immunohistochemical detection of four di fferent antigens (Table 1) using the Avidin-Biotin Complex (ABC) method (Vector Laboratories, CA, USA). Briefly, the sections were depara ffinized, rehydrated and rinsed with tap water. Endogenous peroxidase was quenched by incubating sections in methanol containing 3% H2O2 for 10 min at room temperature, then they were washed with water for 10 min. Antigens were retrieved using epitope demasking (Table 1), and nonspecific binding was inhibited by incubating the sections for 20 min at room temperature with 10% normal horse serum (detection of CD3) or 10% normal goa<sup>t</sup> serum (detection of Iba1, lambda chain or CD20) in Tris-bu ffered saline (TBS) containing 5 mM Tris•HCl (pH 7.6), 136 mM NaCl and 1% bovine serum albumin. Tissue sections were incubated overnight at 4 ◦C with commercial mono- or polyclonal primary antibodies (Table 1), and then washed three times with TBS. Samples were incubated for 30 min at room temperature with horse anti-mouse serum or goa<sup>t</sup> anti-rabbit serum (1:200, Vector Laboratories; Table 1), washed three times with TBS and then incubated with the ABC kit in TBS for 30 min at room temperature.

**Table 1.** Immunohistochemical protocols used to characterize types of inflammatory cell in skin lesion biopsies.


Finally, the sections were incubated for 5 min with the substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO, USA) and washed with TBS and water. After staining for 45 s with hematoxylin, slides were dehydrated and mounted with DPX (Fluka, Sigma, St. Louis, MO, USA). Stained slides were studied under light microscopy (Olympus BH—2) and photographed using a digital camera (Olympus DP—12). Each immunohistochemical staining included a positive control, in which the target antigen was present in the control section and the specific antibody was used (Figure S1); as well as a negative control, in which the primary antibody was omitted.

#### *2.3. Cell Counting and Statistical Analysis*

A total of 80 slides (4 slides per animal, 5 animals per species) were used for cell counting. Cells positive for each immunostained marker were quantified in five fields of each slide at 400× magnification using an image analysis program (Imaging Software NIS-Elements 3.20, Nikon, Tokyo, Japan). Then the mean proportion of stained cells to total cells was averaged across the five fields.

Descriptive and inferential statistics were used to analyze the distribution of four types of inflammatory cells in skin mange lesions in the four species. Data were tested for normality using the Shapiro–Wilk test. Species di fferences in the total number of inflammatory cells in scabies skin lesions were assessed for significance using the non-parametric Kruskal–Wallis and pairwise comparison tests. The percentage of total cells that stained for each of the four inflammatory cell biomarkers (Iba1, lambda chain, CD3 and CD20) was compared within and between each species using a one-way ANOVA. When significant di fferences were found, the Tukey test for multiple comparisons was applied.

As appropriate, data were expressed as the mean and standard deviation, or as the median and interquartile range. Data were analyzed using SPSS 24 for Windows (IBM, Chicago, IL, USA). A significance level of 0.05 was applied.
