title: Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.
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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK. bl349@cam.ac.uk.
authors.forename: Bethan
authors.identifier: 0000-0001-6511-1818
authors.initials: B
authors.lastname: Lloyd-Lewis
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK. fmd32@cam.ac.uk.|School of Pharmacy, The University of Queensland, Brisbane, 4072, Australia. fmd32@cam.ac.uk.
authors.forename: Felicity M
authors.identifier: 0000-0001-9112-118X
authors.initials: FM
authors.lastname: Davis
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 1QR, UK.
authors.forename: Olivia B
authors.initials: OB
authors.lastname: Harris
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK.
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Hitchcock
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK.
authors.forename: Filipe C
authors.initials: FC
authors.lastname: Lourenco
authors.affiliation: Medical Research Council Laboratory for Molecular Biology, Cambridge, CB2 0QH, UK.
authors.forename: Mathias
authors.initials: M
authors.lastname: Pasche
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK. cjw53@cam.ac.uk.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 1QR, UK. cjw53@cam.ac.uk.
authors.forename: Christine J
authors.identifier: 0000-0002-8548-5902
authors.initials: CJ
authors.lastname: Watson
mesh_terms: D000818:Animals; D005260:Female; D005455:Fluorescent Antibody Technique; D021621:Imaging, Three-Dimensional / Q000379:methods; D008321:Mammary Glands, Animal / Q000000981:diagnostic imaging*; D015674:Mammary Neoplasms, Animal / Q000000981:diagnostic imaging* / Q000473:pathology*; D051379:Mice; D018613:Microscopy, Confocal; D061848:Optical Imaging / Q000379:methods
keywords: 3D imaging; Breast cancer; Fluorescence microscopy; Lactation; Light sheet fluorescence microscopy; Mammary gland; Tissue clearing; Two-photon microscopy