Bz 9000 series

Following published protocols (30 (link), 31 (link)), cryosections of human skin, that was obtained from elective plastic surgery, were incubated with immunoapheresis materials or IgG isolated from serum of two EBA patients using protein G columns, of EBA patients for 1 h at 37°C, followed by addition of human PMNs isolated by dextran sedimentation (Carl Roth, Karlsruhe, Germany) of freshly collected, heparinized blood from healthy volunteers (n=3). Afterwards, 0.5 ml of PMNs (2-2.5 × 107 cells), pre-incubated with solvent or parsaclisib at 2, 20, and 200 nM. PMNs, pre-incubated with solvent or parsaclisib at varying concentrations (2, 20, and 200 nM/mL) for 15 min at 37°C, were added onto the skin sections. Slides were further incubated at 37°C for 3 h. After washing, slides were fixed in formalin and H&E-stained. Using ImageJ software (https://imagej.nih.gov/ij/), DES split formation was quantified. The percentage of dermal-epidermal separation (DES) was calculated as length of separation divided by total length of the dermal-epidermal junction (DEJ) on skin section measured on a Keyence microscope (BZ-9000 series, Keyence GmbH, Neu-Isenburg, Germany).

Serial 4-μm-thick sections of SMGs were cut from the block of formalin-fixed and paraffin-embedded tissue and stained with a conventional avidin–biotin complex technique as previously described.^{5 (link)} Antibodies used included anti-CD68 (catalog #ab955; Abcam, Cambridge, MA), anti-CD163 (catalog # NCL-CD163; Leica Biosystems, Nussloch GmbH, Germany), and anti-CD123 (catalog # NCL-L-CD123; Leica Biosystems) mouse monoclonal antibodies; anti-CD11c (catalog #52632; Abcam) and anti-CD21 (catalog #ab75985; Abcam) rabbit monoclonal antibodies and a rabbit anti-MARCO polyclonal antibody (catalog #AP9891A; ABGENT, San Diego, CA). Tissue sections were sequentially incubated with primary antibodies for 2.5 hours then with biotinylated anti-mouse IgG or anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA), avidin–biotin–horseradish peroxidase complex (Vector Laboratories), and 3,3′-diaminobenzidine (Vector Laboratories). Mayer hematoxylin was used for counterstaining. Photomicrographs were obtained using a light microscope equipped with a digital camera (BZ-9000 series; Keyence, Tokyo, Japan).