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

Abstract

BackgroundHigh-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.MethodsMultiple 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.ResultsVarying 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.

Sinus‐like dilatations of the mammary milk ducts, Ki67 expression, and CD3‐positive T lymphocyte infiltration, in the mammary gland of wild European rabbits during pregnancy and lactation

“…However, in veterinary species, welfare considerations are paramount, and this approach may be prohibited because of the use of sedation in pregnant and lactating rabbits. An alternative methodology which would likely yield informative results is high-resolution three-dimensional imaging using tissue clearing, which has recently been described in the mouse mammary gland Lloyd-Lewis et al 2016). A potential strength of our report is that the rabbits were wild and so no impact of artificial husbandry in a farm or laboratory environment is likely.…”

Multiscale activity imaging in mammary gland reveals how oxytocin enables lactation

“…Lacrimal and epididymal tissue was dissected and fixed in NBF for 4-7 h. Pre-stimulation with OT [85 nM (lacrimal and mammary), 850 nM (epididymis), 5-20 min] was performed on live tissue prior to fixation, as indicated. Tissue clearing was performed on fixed tissue using a modified CUBIC protocol (15, 36). Briefly, tissue was immersed in CUBIC Reagent 1A [urea (10% w/w), Quadrol® (5% w/w), triton X-100 (10% w/w), NaCl (25 mM) in distilled water] for 2-3 days at 37°C, washed in PBS and blocked overnight at 4°C in PBS containing normal goat serum (10%) and triton X-100 (0.5%).…”

“…S1). GCaMP6f consists of a circularly permuted green fluorescent protein (GFP), enabling 3D assessment of its expression and lineage-specific localization using an anti-GFP antibody (14) and optimized methods for tissue clearing (15). Genetic recombination in this model was high (Fig.…”

Epithelial plasticity in the mammary gland

“…However, a recent report showed that the enzymatic digestion method used to prepare mammary ducts for 3D imaging can deplete myoepithelial cells and cause structural damage to ducts, and potentially might also deplete rare bi-lineage clones 20 . New methods of micro-dissection or clearing, together with whole-organ 3D imaging analysis and mathematical modeling 8, 12, 13, 21–23 , will undoubtedly provide a clearer picture of mammary gland cell fate during normal development.…”