1. Location, morphology and gene expression of skin CD8α+MHC II+ cells.
Location, morphology and gene expression of skin CD8α+MHC II+ cells. (A) For immunofluorescence analysis, cryostat sections with a thickness of 20 μm were prepared from rainbow trout skin, fixed, and labeled with anti-CD8α (red) and anti–MHC class II (green) Abs, counterstained with Hoechst (blue), and analyzed by confocal microscopy. Arrowheads show double positive cells. Scale bars, 20 μm. (B) For identification of CD8α+MHC II+ cells, total leukocytes from skin were fixed and labeled with anti-CD8α (green) and anti–MHC class II (red) Abs, counterstained with Hoechst (blue), and analyzed by fluorescence microscopy. Scale bars, 5 μm. (C) CD8α+MHC II+ cells from skin were isolated by cell sorting and then incubated onto poly-l-lysine–treated glass slides, fixed, mounted, and analyzed by light microscopy. Scale bar, 5 μm. (D) Myeloid CD8α+MHC II+ cells from skin also were analyzed using a scanning electron microscope. Scale bar, 5 μm. (E) Sorted myeloid skin CD8α+MHC II+ cells were analyzed by real time-PCR. Relative expression of the indicated genes to the endogenous control EF-1α was calculated for each sample, and mean (± SD) values from three independent experiments, including three fish per experiment, were obtained.
Aitor G. Granja et al. J Immunol 2015;195:
Copyright © 2015 by The American Association of Immunologists, Inc.