A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response

• Published in: Histochemistry and cell biology Vol. 144; no. 3; pp. 195 - 208
• Main Authors: Kitazawa, Yusuke, Ueta, Hisashi, Hünig, Thomas, Sawanobori, Yasushi, Matsuno, Kenjiro
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2015; Springer Nature B.V
• Subjects: Animals; Antimetabolites - pharmacology; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bromodeoxyuridine - pharmacology; CD28 Antigens - biosynthesis; Cell Biology; Cell Proliferation - drug effects; Dendritic Cells - drug effects; Dendritic Cells - immunology; Deoxyribonucleic acid; Deoxyuridine - analogs & derivatives; Deoxyuridine - pharmacology; Developmental Biology; DNA; Flow Cytometry; Histology; Immune system; Immunity, Cellular - physiology; Immunohistochemistry; Lymphocytes - immunology; Major Histocompatibility Complex - immunology; Male; Original Paper; Rats; Rats, Inbred Lew; Rodents; S Phase - drug effects; Spleen - cytology; Spleen - immunology; Transcription Factors - biosynthesis; Multicolor immunofluorescence staining; Immunohistology; Flow cytometry; Immunoproliferative response; EdU; T-cell dendritic cell cluster
• ISSN: 0948-6143; 1432-119X
• DOI: 10.1007/s00418-015-1329-z

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.