Standard procedures were used for histology and immunohistochemistry as previously described18 (link) and detailed in the supplemental materials. All routine H&E staining and IHC were conducted using ST5010 autostainer and BondRX immunostainer, respectively (both from Leica, Wetzlar, Germany). Standardized IHC protocol was used for anti-IBA-1 (Wako Chemicals, Richmond, VA) staining. Stained tissue slides were digitized with a P250 pathology slide scanner (Perkin Elmer, Waltham, MA) and analyzed by HALO software (Indica Labs, Corrales, NM).
For human and mouse skin samples, 4 µm microtome sections were collected on slides for H&E and IHC staining. An analysis technique was chosen to minimize the impact of variations in samples since total skin area as well as the proportion of epidermis to dermis can change as the disease progresses. Also, human biopsy collection techniques can result in “edge effects” in the dermis. Thus, for human biopsies, IBA1+ staining was normalized to the length of epidermis evaluated, and data are presented as area (µm2) divided by length (µm). For mouse ear samples, a standard 5 mm length was analyzed for each type of staining, and data are reported as area (mm2 or µm2) for both the H&E and IHC assessment.
For human and mouse skin samples, 4 µm microtome sections were collected on slides for H&E and IHC staining. An analysis technique was chosen to minimize the impact of variations in samples since total skin area as well as the proportion of epidermis to dermis can change as the disease progresses. Also, human biopsy collection techniques can result in “edge effects” in the dermis. Thus, for human biopsies, IBA1+ staining was normalized to the length of epidermis evaluated, and data are presented as area (µm2) divided by length (µm). For mouse ear samples, a standard 5 mm length was analyzed for each type of staining, and data are reported as area (mm2 or µm2) for both the H&E and IHC assessment.