Kitazawa Yusuke, Ueta Hisashi, Hünig Thomas, Sawanobori Yasushi, Matsuno Kenjiro
Department of Anatomy (Macro), Dokkyo Medical University, 880 Kitakobayashi, Mibu, Tochigi, 321-0293, Japan.
Histochem Cell Biol. 2015 Sep;144(3):195-208. doi: 10.1007/s00418-015-1329-z. Epub 2015 May 15.
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.
免疫反应通常伴随着抗原呈递以及抗原特异性淋巴细胞的增殖和分化（免疫增殖），但是在组织切片上对这些事件进行原位分析非常困难。我们开发了一种新的方法，使用胸腺嘧啶类似物5-乙炔基-2'-脱氧尿苷（EdU）进行免疫组织学和流式细胞术的同步多色免疫荧光染色。由于使用点击化学的叠氮化物染料体积小且无需DNA变性步骤，EdU染色能够在单细胞表面进行至少四种颜色的免疫荧光染色，包括两种不同的标记物，这是标准的5-溴-2'-脱氧尿苷方法无法做到的。通过使用两种大鼠模型，成功检测的参数包括分化抗原簇，其中包括各种免疫细胞的表型和功能标记物、组织相容性复合体抗原，甚至一些核转录因子。增殖细胞可以进一步分选并用于逆转录聚合酶链反应分析。因此，该方法能够在淋巴器官的不同区域对免疫增殖反应进行功能性原位时间动力学分析，流式细胞术对其进行了定量确认。
