In all, 330,000 HDF per HSE were embedded in a collagen matrix containing rat tail collagen type I (Corning), 10× DMEM (Thermofisher) and sodium bicarbonate (Gibco/Invitrogen), filled into six-well-culture inserts (Thermofisher) and placed in deep six-well culture plates (Thermofisher). After 2 h of polymerisation at 37 °C, HSEs were equilibrated in DMEM medium supplemented with 10% foetal bovine serum (Sigma) and placed at 37 °C, 5% CO2. After 3 days, 150,000 keratinocytes were seeded on top and submerged for 7 days in DMEM medium supplemented with 10% foetal bovine serum and growth factors (EGF, isoproterenol, hydrocortisone). Then the inserts were placed at the air–liquid interface for 7 days during which period the medium was supplemented with 1201.
HSEs were fixed in 10% formalin before embedding in paraffin and cutting into 5-µm-thick sections. Haematoxylin and eosin staining was performed using a standard protocol. Immunohistochemistry staining was performed using a standard protocol and the following primary antibodies: fillagrin (Abcam, ab17808), loricrin (Abcam, ab24722), keratin 10 (Abcam, ab9026) and the following secondary antibodies: α-mouse Alexa 488 (Abcam, ab150113) and α-rabbit Alexa 546 (Life technologies, A11010).
HSEs were fixed in 10% formalin before embedding in paraffin and cutting into 5-µm-thick sections. Haematoxylin and eosin staining was performed using a standard protocol. Immunohistochemistry staining was performed using a standard protocol and the following primary antibodies: fillagrin (Abcam, ab17808), loricrin (Abcam, ab24722), keratin 10 (Abcam, ab9026) and the following secondary antibodies: α-mouse Alexa 488 (Abcam, ab150113) and α-rabbit Alexa 546 (Life technologies, A11010).