Double sided adhesive tape

Epidermal sheets were prepared as previously described (Mohammed et al., 2016 (link)). Briefly, skin fat was mechanically removed and subsequently mounted on microscopy slides that had been precoated with double-sided adhesive tape (3M, St. Paul, MN). Slides were incubated in 10 mM EDTA at 37 °C for 45–90 minutes. The dermis was peeled away from the epidermis with standard forceps. Skin-whole mounts were prepared from skin samples embedded in optimal cutting temperature compound, and 8 μm transverse skin slice sections were prepared. Epidermal sheets and skin-whole mounts were fixed in 4% paraformaldehyde at room temperature for 30 minutes and blocked for 1 hour at room temperature in PBS buffer containing 0.1% tween-20, 2% BSA, and 2% rat serum. Immunostaining of skin samples was done overnight in PBS containing 0.1% tween-20 and 0.5% BSA. Skin samples were stained with anti-EpCAM(G8.8)-AF647, anti-A/I-E/MHCII-AF488, and anti-Langerin(CD207)-phycoerythrin, followed by anti-phycoerythrin-AF555 and DAPI. Images were captured on an IX83 fluorescent microscope (Olympus, Tokyo, Japan) using a ×10 objective; image analysis was performed using cellSens Dimension software (Olympus).

SC samples were collected from the lesional and nonlesional areas in the leg, arm, abdomen, or back skin of patients with NE and the same skin areas of healthy volunteers using Scotch^® tape (3M, St Paul, MN, USA). For western blot analysis, the 1.8 cm × 5 cm Scotch^® tape was placed on the lesional and nonlesional skin of patients with NE (mean age ± standard error of the mean (SEM), 51.3 ± 2.72 years; age range, 27–71 years; 12 females and 12 males) or skin of healthy volunteers (48.9 ± 2.12 years; age range, 36–59 years; 10 females and 5 males) for 3 minutes, applying pressure during the last minute, and then attached and detached 10 times. For immunofluorescence staining, the SC samples were obtained using the 0.84 cm × 3 cm double-sided adhesive tape (3M) on the slide by placing the tape-adhered slide on the skin of patients with NE or healthy volunteers for 3 minutes, applying pressure during the last minute. Tapes and slides containing SC tissues were stored at –20°C until use.