Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.
The present study investigated the potential involvement of interferon‐γ (IFN‐γ)‐producing cells in the pathogenesis of oral lichen planus (OLP). On biopsies from 10 OLP patients, an in situ hybridization technique was employed to determine the topographic al distribution of cells expressing IFN‐γ mRNA. It was estimated that approximately 1% or fewer lesional cells were IFN‐γ mRNA‐positive. These cells were mainly encountered lining the basal membrane in a majority of the patients, or were in a few cases circumscribing the infiltrate, but were more seldon localized to the center of the lesion. A slightly higher, but not statistically significant number of phytohemagglutinin (PHA)‐induced IFN‐γ‐producing cells, in vitro, was found in blood from 11 other OLP patients compared with blood from matched controls. Equal concentration of IFN‐γ in supernatants from PHA‐stimulated blood cells were detected in the two groups. Similarly, the IFN‐γ response towards C. albicans was alike in OLP and in healthy control (HC) blood cells, indicating normal immunological memory function in the OLP patients. A small set of cells with spontaneous IFN‐γ production was found in OLP and in HC peripheral blood. The data suggest that T‐lymphocyte activation and cytokine production act locally and are not reflected in peripheral blood. The localization of the IFN‐γ mRNA‐positive cells indicates that the antigenic peptides are presented at the periphery of the mononuclear cell infiltrate. Furthermore, the low frequency of IFN‐γ mRNA‐positive cells in the lesions suggests that the disease is maintained by a small number of antigen‐specific T cells.
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