Figure 3
Imaging modality to whole animal in vivo fluorescence. (a) Immunofluorescent staining showing the expression of basal epithelial marker (K5 and K14) in mammary organoids generated from Rosa26 tdTomato mammary gland. The Red fluorescence is tdTomato endogenous fluorescence (Scale bar = 100 µm). (b) Schematic showing imaging modalities to whole gland imaging and whole animal imaging. Animals were anaesthetized after 4 weeks post transplantation and imaged for tdTomato fluorescence at the left side (site for transplantation) and also checked for mammary fatpad imaging through ultrasound imaging. The mammary glands were dissected out and checked for the endogenous tdTomato fluorescence to visualize ductal trees, and also performed whole-gland immunostaining to check tdTomato-derived mammary epithelial cells. (c) In vivo whole animal fluorescence (Xenogen IVIS fluorescence imager) reveals tdTomato expression at the site of transplantation. The right side was used as control (n = 5 pregnant mice). The Radiant efficiency scale in the whole animal fluorescence is expressed as (p/sec/cm2/sr)/(µW/cm2). Color scale: Min = 1.00e8 and Max = 1.00e9. (d) Ultrasound imaging reveals the formation of mammary fat pad and complete absence of the lymph node (LN) confirming efficient mammary fat pad clearing and the ability of mammary organoids to reconstitute mammary gland (n = 5 pregnant mice). (e) Tissue clearing enables 3D confocal imaging to visualize mammary ductal tree formation. The mammary glands were surgically dissected after 4 weeks post transplantation (virgin) and incubated in Fructose-Glycerol clearing Solution (FGCS) for 24 h, making the whole tissue translucent for efficient imaging. Tissue clearing can also be performed for 3–7 days (Pregnant mice) depending on the stage of mammary gland development. Confocal microscopy of the whole gland was subsequently imaged to check for the presence of ductal trees, and the endogenous tdTomato fluorescence confirms organoids are able to regenerate mammary gland ductal tree in vivo (Scale bar = 1 mm).
