Alexa fluor 594 anti human igg

Injected skin specimens without mechanical stress area were frozen in OCTcompound (SAKURA, Tokyo, Japan) by liquid nitrogen and kept at −80 °C until use. Skin samples were sliced into 4 μm sections. Skin specimens were blocked with Tris-bufferd saline (TBS) containing 1% albumin from bovine serum and 1 mM CaCl₂ for 30 min at room temperature, followed by incubation with primary antibodies in blocking solution for 1 h at room temperature. After three time washes in TBS-Ca, the skin sections were incubated with appropriated conjugated secondary antibodies diluted 1:400 in the blocking solution for 1 h at room temperature. The following mAbs were used for primary antibodies: a mouse anti-Dsg1 mAb (27B2, 1:20 dilution, Abcam, Cambridge, UK), a mouse anti-PG mAb (15F11, 1:1000 dilution, Sigma-Aldrich), a mouse anti-Dsc1 mAb (U100, undiluted, PROGEN Biotechnik GmbH, Heidelberg, Germany), and a mouse anti- Dsg3 mAb (G194, undiluted, Acris Antibodies GmbH, Herford, Germany). Double staining of deposited IgG and adhesion molecules was performed with Alexa Fluor 488 anti-mouse IgG (Invitrogen) and Alexa Fluor 594 anti-human IgG (Invitrogen) as secondary Abs. Immunofluorescence was observed at ×1000 magnification by Nikon laser confocal microscope A1R (Nikon, Tokyo, Japan).