title: Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.
abstract: Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyﬂuorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.
authors.affiliation: Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Institute of Diabetes Obesity and Metabolism, Institute for Regenerative Medicine, Department of Pediatrics, University of Pennsylvania-School of Medicine, USA.
authors.forename: Alex H
authors.initials: AH
authors.lastname: Tuttle
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Rankin
authors.forename: Monica
authors.initials: M
authors.lastname: Teta
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sartori
authors.forename: Geneva M
authors.initials: GM
authors.lastname: Stein
authors.forename: Gina J
authors.initials: GJ
authors.lastname: Kim
authors.forename: Cristina
authors.initials: C
authors.lastname: Virgilio
authors.forename: Anne
authors.initials: A
authors.lastname: Granger
authors.forename: Di
authors.initials: D
authors.lastname: Zhou
authors.forename: Simon H
authors.initials: SH
authors.lastname: Long
authors.forename: Alisa B
authors.initials: AB
authors.lastname: Schiffman
authors.forename: Jake A
authors.initials: JA
authors.lastname: Kushner
mesh_terms: D000818:Animals; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000032:analysis; D005260:Female; D007065:Idoxuridine / Q000032:analysis*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010179:Pancreas / Q000737:chemistry; D013936:Thymidine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: C012244:5-chloro-2'-deoxyuridine; D007065:Idoxuridine; D013936:Thymidine; D003857:Deoxyuridine