期刊
BREAST CANCER RESEARCH
卷 18, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s13058-016-0754-9
关键词
Mammary gland; Lactation; Breast cancer; Tissue clearing; 3D imaging; Fluorescence microscopy; Light sheet fluorescence microscopy; Two-photon microscopy
类别
资金
- Medical Research Council (MRC) [MR/J001023/1]
- National Health and Medical Research Council CJ Martin Biomedical Fellowship [GNT1071074]
- Wellcome Trust [105377/Z/14/Z]
- MRC [MR/K011014/1]
- Cancer Research UK
- MRC-LMB [MC_U105178788]
- Wellcome Trust [105377/Z/14/Z] Funding Source: Wellcome Trust
- MRC [MR/K011014/1, MR/N022963/1, MR/J001023/1, MC_U105178788] Funding Source: UKRI
- Medical Research Council [MC_U105178788, MC_PC_12009, MR/K011014/1, MR/J001023/1, MR/N022963/1] Funding Source: researchfish
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.
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