Human skin samples
Skin biopsies were taken from body donors received by the Institute of Anatomy and Cell Biology, Ludwig-Maximilian-Universität München, Germany. Body donors had given written informed consent for use of tissue samples for research purposes. The procedure was approved by the Ethics Committee of the Faculty of Medicine (project no. 249-12). Only samples from body donors deceased for <24 h were used. Each epidermis piece ~4 cm² was excised from the shoulder region and divided into ~1 cm² pieces.
Either 50 µl of the PLC -inhibitor U-73122, 4 µM in DMSO/PBS 1:50 or vehicle were injected intra-epidermally, followed by floating incubation in 5 ml Dulbecco’s modified Eagle’s medium, containing 10 % FCS, 50 U/ml penicillin and 50 mg/ml streptomycin, with the epidermis facing upwards at the liquid-air-interface without additional support at 95 % humidity, 5 % CO2 and 37 °C. After 1 h incubation, either PV2 or IgG obtained from healthy volunteers, purified for experimental use by protein A affinity chromatography as described previously was injected. Injections were performed using a 0.01-1 ml syringe (B.Braun, Germany), and a 0.3x12 mm BL/LB 30Gx1/2” needle (B.Braun, Germany). After 24 h, shear stress was applied using a rubber piece. The skin pieces were cut and processed for either hematoxylin and eosin (H&E) or immunostaining.