Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods
Authors
Lloyd-Lewis, Bethan
Davis, Felicity M
Harris, Olivia
Hitchcock, Jessica
Lourenco, Filipe C
Pasche, Mathias
Publication Date
2016Journal Title
Breast Cancer Research
ISSN
1465-5411
Publisher
BioMed Central
Language
English
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Lloyd-Lewis, B., Davis, F. M., Harris, O., Hitchcock, J., Lourenco, F. C., Pasche, M., & Watson, C. (2016). Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Research https://doi.org/10.17863/CAM.6345
Abstract
$\textbf{Background}$: 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.
$\textbf{Methods}$: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3DISCO, SeeDB, CUBIC and PACT. Using confocal, twophoton and light sheet microscopy, their compatibility with wholemount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed.
$\textbf{Results}$: 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 wholemount 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.
$\textbf{Conclusions}$: 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 $\textit{in situ}$, and will significantly enhance our understanding of both normal and pathological mammary gland development.
Keywords
mammary gland, lactation, breast cancer, tissue clearing, 3D imaging, fluorescence microscopy, light sheet fluorescence microscopy, two-photon microscopy
Sponsorship
This work was supported by a grant from the Medical Research Council (MRC) program grant no. MR/J001023/1 (B.L-L. and C.J.W.). F.M.D. was funded by a National Health and Medical Research Council CJ Martin Biomedical Fellowship (GNT1071074). O.B.H. was funded by a Wellcome Trust PhD Studentship (105377/Z/14/Z). J.R.H was funded by an MRC research grant no. MR/K011014/1. F.C.L. was funded by Cancer Research UK and M.P. was funded by the MRC-LMB (MC_U105178788).
Funder references
MRC (MC_PC_12009)
MRC (MR/K011014/1)
MRC (MR/J001023/1)
MRC (MR/N022963/1)
WELLCOME TRUST (105377/Z/14/Z)
Embargo Lift Date
2100-01-01
Identifiers
This record's DOI: https://doi.org/10.17863/CAM.6345
This record's URL: https://www.repository.cam.ac.uk/handle/1810/261176