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.