Immunohistochemistry

Perilesional skin biopsy specimens from 13 patients with BP before the start of treatment were obtained. Tissues were fixed in paraformaldehyde, embedded in paraffin, and sectioned. Immunostaining with a mouse monoclonal anti-human NLRP3 antibody (clone Nalpy3-b, Enzo Life Sciences, Villeurbanne, France) or a mouse monoclonal anti-human CD163 antibody (Clone MRQ26, Roche Diagnostics, Meylan, France) was performed using routine methods with a biotinylated anti-mouse secondary antibody (BA-2000; Vector Laboratories, Burlingame, USA) and the ABC-peroxidase complex (Vector Laboratories, Burlingame, USA) with diaminobenzidine-H₂O₂ used as the chromogen for detection. In order to identify cells positive for both NLRP3 and CD163, immunohistochemistry for both markers was performed on 4 μm-thick serial BP skin section. Virtual slide images for NLRP3 and CD163 staining were taken using the VS120 Virtual Slide Microscope (Olympus France, Rungis, France). Virtual slide images corresponding to NLRP3 and CD163 staining were opened at the same time with OlyVIA software (Olympus France, Rungis, France) and synchronized for analysis.