Anti aquaporin 3

After 24-h treatments, EpiDerm-FT™ cultures were fixed in 10% formalin and later preserved by paraffin embedding. Sections (5 µm) were stained with hematoxylin and eosin (H&E) for evaluation of tissue morphology. Immunohistochemistry (IHC) was performed using anti-aquaporin-3 (AbCam; Cambridge, UK) and anti-keratin-10 (EMD Millipore; Billerica, MA). Secondary antibodies used were anti-rabbit-Alexa Fluor-488 and anti-mouse-Alexa Fluor-594 (Jackson Immuno Research Labs; West Grove, PA). Antibody specificity was confirmed by staining tissues with secondary antibodies only. Stained sections were viewed using an Olympus BX-41 microscope (Center Valley, PA) equipped with CCD camera (Hamamatsu Photonics K.K.; Shizuoka Pref., Japan) and using MetaMorph^® software (Molecular Devices, Sunnyvale, CA).

Dorsal skin tissue was lysed in RIPA lysis and extraction buffer (Thermo Scientific, Rockford, IL, USA), containing a protease inhibitor cocktail (cOmplete™; Roche, Mannheim, Germany). Anti-aquaporin 3, anti-TSLP (all from Abcam), anti-filaggrin, anti-cytokeratin 14, anti-p-extracellular-signal-regulated kinase (ERK), anti-ERK, anti-p-AKT, anti-AKT (all from Cell Signaling Technology, Danvers, MA, USA), anti-TLR2, anti-MyD88, anti-TRAF6 (all from Invitrogen), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used. Western blot analysis was performed according to a previously published method [31 (link)].