Abstract
Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1329-z) contains supplementary material, which is available to authorized users.
Highlights
Immune responses induce activation and clonal expansion of antigen-specific lymphocytes with active DNA synthesis for cell division
EdU staining correlates well with BrdU staining in immunohistology
The Click-iT® kit resulted in intense and clear EdU staining with a low signal-to-noise ratio for all fluorochromes tested: Alexa-488, -594, and -647
Summary
Immune responses induce activation and clonal expansion of antigen-specific lymphocytes with active DNA synthesis for cell division. DCs form a cluster with antigen-specific lymphocytes and induce immunoproliferation, i.e., their differentiation and proliferation within the cluster (Matsuno et al 1989; Saiki et al 2001; Ueta et al 2008). We have long studied immunoproliferative responses in situ by using a multicolor immunoenzyme staining analysis (Matsuno et al 1989, 2010; Saiki et al 2001; Ueta et al 2008) for a thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), and other parameters. Because enzymedeveloped color dyes tend to interfere with the following immunostaining step, only two-color analysis prior to BrdU staining could be performed at best and detection of two different markers on a single-cell surface was very difficult. BrdU immunofluorescence staining is problematic, because it requires DNA denaturation steps
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