Localisation of CD123+ pDC in uninfected and HIV-1-infected thymus tissue. Immunohistochemistry was performed on fresh frozen thymus sections. Tissue sections were examined using an Olympus BX50 microscope (Olympus, Centre Valley, PA) and images captured using a ProSeries 3CCD camera (SciTech). Sections were imaged with a 20x objective and montages were created and composite images were colour deconvoluted using H-DAB filter in ImageJ (National Institutes of Health, Bethesda, MD). The threshold was set for 3,3'-Diaminobenzidine-positive cells (red). The medulla (M) was identified by the presence of Hassall's corpuscles (HC, open arrow head), increased HLA-DR expression, high CD40 expression and the presence of CD83+ medullary DC. (A) In thymus tissue from uninfected human donors the medulla and cortex [C] included cells expressing high levels of CD123. (B) The distribution of CD123+ cells in thymic grafts from day 7 HIV-1BaL-infected SCID-hu-thy-liv mice was similar to normal thymus tissue. (C) Sections were labelled with isotype control or p24 antibody and processed for immunohistochemistry using DAB. The cortical regions were imaged at an original magnification of 20x and a montage created before processing for colour deconvolution using ImageJ. The threshold was set on the DAB channel, particles analysed using ImageJ, and particle masks were then overlayed onto the original montaged image. Scale bar is 200 μm.