Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods

Lloyd-Lewis, Bethan, Davis, Felicity M., Harris, Olivia B., Hitchcock, Jessica R., Lourenco, Filipe C., Pasche, Mathias and Watson, Christine J. (2016) Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Research, 18 1: 127-127. doi:10.1186/s13058-016-0754-9


Author Lloyd-Lewis, Bethan
Davis, Felicity M.
Harris, Olivia B.
Hitchcock, Jessica R.
Lourenco, Filipe C.
Pasche, Mathias
Watson, Christine J.
Title Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods
Journal name Breast Cancer Research   Check publisher's open access policy
ISSN 1465-542X
1465-5411
Publication date 2016-12-13
Year available 2016
Sub-type Article (original research)
DOI 10.1186/s13058-016-0754-9
Open Access Status DOI
Volume 18
Issue 1
Start page 127
End page 127
Total pages 17
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Abstract High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated.
Formatted abstract
Background: High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated.

Methods: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed.

Results: Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue.

Conclusions: The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.
Keyword 3D imaging
Breast cancer
Fluorescence microscopy
Lactation
Light sheet fluorescence microscopy
Mammary gland
Tissue clearing
Two-photon microscopy
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID MC_PC_12009
MC_U105178788
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Pharmacy Publications
 
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