Ab85679

In 8-well chamber slides, KCs (1 × 10⁴ cells/well) were seeded and treated with the positive control agent (1.8 mM calcium) or 0.05% H.ECM™ liposome when they reached over 80% confluence and incubated for 24 h. For immunofluorescence, the 8-well chamber slide containing treated KC cells and human skin cryosections (6 μm-thick) were fixed at room temperature (20–25 °C) with 4% (w/v) paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 15 min. Post-fixation, the chamber or cryosections were washed with phosphate-buffered saline (PBS)-T (PBS containing Triton X-100; DAEJUNG, Busan, KR) three times, followed by incubation with anti-filaggrin (ab81468; Abcam) or nti-loricrin (ab85679; Abcam) antibodies overnight at 4 °C. After washing three times with PBS-T, samples were incubated with the FITC-labeled secondary fluorescent antibody (Goat pAb to Rb IgG-FITC, ab6717; Abcam) for 2 h at room temperature to bind primary antibodies. Finally, the cells and tissue specimen were fixed using the fixation solution (VECTASHIELD^® mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA). All fluorescence images were acquired using a laser-scanning microscope (LSM 700, Carl Zeiss, Jena, Germany), and fluorescence intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

To investigate the effect of the GTB1 on AD, the lesion area of the test animal tissue was fixed in 10% neutral buffered formalin at the end of the experiment. After step-by-step dehydration using a high-concentration ethanol solution (starting from a low concentration), paraffin blocks were prepared. Each tissue block was cut to a thickness of 5 μm to facilitate attachment to the slide, and each tissue slide was deparaffinized with xylene and dehydrated using alcohol. To evaluate epidermal thickening, tissue slides were stained with hematoxylin and eosin (H&E). Mast cells in the skin were stained with toluidine blue (TB). Some skin sections were stained for anti-filaggrin (1 : 250, GTX37695, Gene Tex) and anti-loricrin (1 : 2000, ab85679, Abcam) antibodies in accordance with the manufacturer's instructions. The slices were washed with PBS, dehydrated, and mounted in a Permount mounting medium (SP15-100, Thermo Scientific). All stained tissue slides were photographed using a slide scanner (Pannoramic MIDI; 3DHISTECH Ltd, Budapest, Hungary) and observed using Case Viewer software. All histological examinations were analyzed in 3 sections/animal slices.