Real-Time Visualization of Macromolecule Uptake by Epidermal Langerhans Cells in Living Animals Rachel E. Frugé, Colleen Krout, Ran Lu, Hironori Matsushima,

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Real-Time Visualization of Macromolecule Uptake by Epidermal Langerhans Cells in Living Animals Rachel E. Frugé, Colleen Krout, Ran Lu, Hironori Matsushima, Akira Takashima Journal of Investigative Dermatology Volume 132, Issue 3, Pages 609-614 (March 2012) DOI: 10.1038/jid.2011.385 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Direct visualization of macromolecule endocytosis by epidermal Langerhans cells. Anesthetized wild-type BALB/c mice received subcutaneous injection of 70kDa FITC-dextran probes into the ear skin. At 0 (a), 24 (b), and 48hours (c) after the injection, the mice were placed under a confocal microscope to record FITC fluorescence signals in the ear skin. Data shown are compiled x-y plane fluorescence images in the indicated z-axis depth ranges from the skin surface, i.e., stratum corneum. Bar=200μm. Journal of Investigative Dermatology 2012 132, 609-614DOI: (10.1038/jid.2011.385) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identity of the epidermal cells that have incorporated FITC-dextran (DX) probes. BALB/c mice received subcutaneous injection of 70kDa FITC-DX probes into ear skin. At 36hours after the injection, the ear samples were harvested for immunofluorescence staining of the EDTA-separated epidermal sheets with Alexa Fluor 546-conjugated mAb against Langerin (a) or against major histocompatibility complex (MHC) class II molecules (b, c). Bar=50μm. Journal of Investigative Dermatology 2012 132, 609-614DOI: (10.1038/jid.2011.385) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Association of internalized dextran (DX) probes with major histocompatibility complex (MHC) II molecules in Langerhans cells. I-Aβ-enhanced green fluorescent protein (EGFP) knock-in mice (a, b) and Langerin-EGFP-diphtheria toxin receptor knock-in mice (c, d) received subcutaneous injection of 70kDa Alexa Fluor 546 (AF546)-DX probes into the ear skin. At 32hours, the mice were examined under a confocal microscope. Data shown are compiled x-y plane images within the epidermal component (i.e., 0–19μm z-axis depth range from the skin surface). Bar=50μm. Journal of Investigative Dermatology 2012 132, 609-614DOI: (10.1038/jid.2011.385) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions