Pubertal ductal morphogenesis: isolation and transcriptome analysis of the terminal end bud

Morris, J. S. and Stein, T. (2017) Pubertal ductal morphogenesis: isolation and transcriptome analysis of the terminal end bud. In: Martin, F., Stein, T. and Howlin, J. (eds.) Mammary Gland Development: Methods and Protocols. Series: Methods in Molecular Biology (1501). Springer Publishing Company: New York, pp. 131-148. ISBN 9781493964734 (doi:10.1007/978-1-4939-6475-8_5)

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The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole tissue sections are being analysed. Here we describe a method to enzymatically separate TEB and ducts respectively from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has recently allowed us to identify the glycoprotein fibulin-2 (FBLN2) and its binding partner versican (VCAN) as novel TEB-associated proteins. We further include protocols for the processing of mammary tissue into paraffin and immunohistochemical/-fluorescent staining for verification and localisation of protein expression in the mammary tissue at different developmental time points.

Item Type:Book Sections
Glasgow Author(s) Enlighten ID:Stein, Dr Torsten and Morris, Professor Joanna
Authors: Morris, J. S., and Stein, T.
Subjects:Q Science > Q Science (General)
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > School of Veterinary Medicine
Publisher:Springer Publishing Company
Published Online:29 October 2016

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