Live Confocal Microscopy of Oligonucleotide Uptake by Keratinocytes in Human Skin Grafts on Nude Mice  Paul J. White, Rhys D. Fogarty, Ingrid J. Liepe, George A. Werther, Christopher J. Wraight  Journal of Investigative Dermatology  Volume 112, Issue 6, Pages 887-892 (June 1999) DOI: 10.1046/j.1523-1747.1999.00593.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histologic confirmation of successful grafting of human skin on to athymic mice. Grafts were removed at 42 d postgrafting and processed as described in Materials and Methods, as were sections of human skin prior to grafting. (a) Haematoxylin and eosin stained section of human skin prior to grafting. (b) Haematoxylin and eosin stained section of human skin graft showing athymic mouse skin, junction between mouse and human skin, and human skin only from right to left. An arrow indicates the graft margin. Scale bars: 50 μm. Journal of Investigative Dermatology 1999 112, 887-892DOI: (10.1046/j.1523-1747.1999.00593.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Intradermal injection of ODN into human skin grafts results in widespread uptake of ODN into epidermal keratinocytes. Live confocal images were obtained 4 h after intradermal injection of 10 nmol ODN into human skin grafts on nude mice. (a) Corneocytes containing ODN; (b,c) suprabasal and basal keratinocytes containing nuclear ODN; (d) lower magnification image of basal keratinocytes containing nuclear ODN. Scale bars: (a–c) 20 μm; (d) 40 μm. (e–h) Fixed 5 μm sections removed from same graft viewed using LCM; (e, f) uptake of ODN into nuclei of epidermal keratinocytes from the stratum granulosum to the stratum basale; (g) reduced nuclear uptake of ODN into keratinocytes of mouse skin adjacent to the human skin graft; (h) lack of nuclear uptake into keratinocytes of human skin graft receiving intradermal injection of a solution containing 10 nmol dA and 10 nmol propynyl dC. Scale bars: (e–h) 50 μm. Journal of Investigative Dermatology 1999 112, 887-892DOI: (10.1046/j.1523-1747.1999.00593.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Tape stripping results in uptake of ODN into keratinocytes in human skin grafts. ODN was applied to grafts (5 nmol) which had been tape stripped as described in Materials and Methods. Grafts were then viewed using LCM (except in d). (a) Keratinocytes of the granular layer exhibiting largely cytoplasmic ODN; (b, c) suprabasal and basal keratinocytes containing nuclear ODN; (d) fixed section showing uptake of ODN into basal keratinocytes of graft viewed; (e) cytoplasmic uptake of single nucleotides (dA and propynyl dC) into human skin graft keratinocytes. Scale bars: (a–c) 20 μm; (d) 40 μm; (e) 20 μm. Journal of Investigative Dermatology 1999 112, 887-892DOI: (10.1046/j.1523-1747.1999.00593.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Topical application results in little or no nuclear penetration and localization of ODN in keratinocytes in human skin grafts. ODN or ODN/Tfx-50 complexes (50 μl) were topically applied to human skin grafts for 4 h prior to imaging. (a) Application of ODN alone (500 nmol) resulted in localization of the ODN in the stratum corneum; (b) application of ODN/Tfx-50 complexes (500 nmol ODN, 50 μg per ml Tfx-50) allow for penetration of ODN into the epidermal keratinocytes of small regions in human skin grafts; (c) images of fixed sections from skin grafts after topical ODN/Tfx-50 application showing predominant images obtained; localization of the ODN in the stratum corneum. Scale bars: (a, b) 20 μm; (c) 40 μm. Journal of Investigative Dermatology 1999 112, 887-892DOI: (10.1046/j.1523-1747.1999.00593.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Internalized FITC-ODN is intact after 24 h in vitro. Normal human keratinocytes were incubated with 1 μM FITC-ODN and 2 μg per ml Tfx-50 for 24 h. Cells were lysed and ODN detected by Western blotting using an antifluorescein antibody. Lanes 1 and 2, cells transfected without lipid; lanes 3 and 4, lanes transfected with Tfx-50 (T). Journal of Investigative Dermatology 1999 112, 887-892DOI: (10.1046/j.1523-1747.1999.00593.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions