In Situ Analysis of Cell Populations: Long-Term Label-Retaining Cells

  • Rodrigo Fernandez-Gonzalez
  • Irineu Illa-Bochaca
  • Dawne N. Shelton
  • Bryan E. Welm
  • Mary Helen Barcellos-HoffEmail author
  • Carlos Ortiz-de-Solorzano
Part of the Methods in Molecular Biology™ book series (MIMB, volume 621)


The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available.

Key words

Bromodeoxyuridine labeling Osmotic pump 3D reconstruction Nuclear morphology Spatial analysis 



This work was supported by a predoctoral fellowship to RFG from the Department of Defense Breast Cancer Research Program (DAMD 17-03-1-0594), grants from the same institution to COS (DAMD 17-00-1-0227 and DAMD 17-00-1-0306), a grant to BEW from the National Cancer Institute (CA 8424306) and a grant to MHBH funded by the National Institute of Environmental Health Sciences and the National Cancer Institute (U01 ES012801).


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Rodrigo Fernandez-Gonzalez
    • 1
  • Irineu Illa-Bochaca
    • 2
  • Dawne N. Shelton
    • 3
  • Bryan E. Welm
    • 3
  • Mary Helen Barcellos-Hoff
    • 2
    Email author
  • Carlos Ortiz-de-Solorzano
    • 4
  1. 1.Development Biology ProgramSloan-Kettering, Institute, Memorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Langone School of MedicineNew York UniversityNew YorkUSA
  3. 3.Department of SurgeryUniversity of Utah, Huntsman Cancer InstituteSalt Lake CityUSA
  4. 4.Morphology and Imaging Group and Cancer Imaging Laboratory, Center for Applied Medical ResearchUniversity of NavarrePamplonaSpain

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