In vivo confocal fluorescence images of MHC-II⁺ cells in the mouse ear reveal two morphologically distinct cutaneous DC populations in superficial epidermal (a) and deeper dermal (b) layers of the tissue. In (b) dermal vessels are labeled with Alexa488-conjugated αCD31. (c-f) Ex vivo whole-mount histology confirms in vivo observations. Excised ears were split and processed; epidermis was stained with αMHC-II (c) and αlangerin (d) to confirm the identification and morphology of Langerhans cells. Image overlay (e) confirms the dual staining. (f) DCs in the dermis were stained with αMHC-II (red), and their distribution relative to αCD31-labeled vessels (green) is shown. These findings were confirmed in 10 and 20 mice for in vivo confocal and whole-mount imaging, respectively. The field of view in all images is 380 μm × 380 μm.

In vivo confocal fluorescence images of an EMT6 tumor 2 h after intradermal injection with fluorophore-conjugated αMHC-II and αCD31. (a) MHC-II⁺ cells (red) visualized at a depth of ∼ 40 μm in the tumor with morphology consistent with that of Langerhans cells. No CD31-positive vasculature is observed at this depth. (c) Positively stained cells (red) at a depth of ∼ 80 μm in the tumor in the presence of a highly vascularized tumor microenvironment. The images (b) and (d) are expanded views of the region of interest indicated by the white box superimposed on (a) and (c), respectively. Imaging experiments were performed in 4 tumor-bearing mice, and the results were found to be reproducible. The field of view in images (a) and (c) is 800 μm × 800 μm, while that in (b) and (d) is 200 μm × 200 μm.