Influence of whole-mount preparation and efficiency of labelling on lineage tracing outcomes.
a Whole-mount three-dimensional (3D) confocal image of an intact ductal portion immunostained for Keratin 5 obtained using a protocol that does not include any enzymatic digestion (described in Rios et al. [
4]).
Scale bar = 50 μm.
b Whole-mount 3D confocal image of a ductal portion (8-week-old FVB/N mouse) immunostained for Keratin 5 (
green) and E-cadherin (
red), using the protocol described by Wuidart et al. for enzymatic digestion of mammary glands [
1]. The mammary portions were subsequently fixed in 4% paraformaldehyde (PFA) for 2 h at 4 °C, and processed as described in Rios et al. [
4] for immunostaining and confocal imaging.
c Image of an enlargement from (
b) showing the myoepithelial layer immunostained for Keratin 5. Note the paucity of myoepithelial cells and their altered shape, which is no longer elongated.
Scale bars = 100 μm (
b) and 30 μm (
c).
d Schematic panels showing normal contacts between one myoepithelial cell (
myo, in
green) and multiple luminal cells (
lum, in
red) in intact breast tissue (
left), and loss of cell-cell contacts between the myoepithelial (in
green) and luminal cells (in
red) after enzymatic digestion (
right).
e Whole-mount 3D confocal image (from Fig.
1d, Rios et al. [
4]) of a duct in a K5rtTA-IRES-GFP mammary gland immunostained for E-cadherin (in
blue).
f enlargement from (
e), showing a mask (in
red) applied to one myoepithelial cell (GFP channel using Imaris software) and the luminal layer labelled with E-cadherin (in
blue).
g Image showing the mask of the myoepithelial cell and the masks of all luminal cells in direct contact with the myoepithelial cell. Note: the mask was applied to a representative 70 μm myoepithelial cell.
Scale bars = 50 μm (whole-mount
e) and 10 μm (enlargements
f,
g).
h, i Whole-mount 3D confocal images of ducts in a K5-KI/R26R-tdTomato mammary gland, showing either a high (
h) or low (
i) degree of labelling in the same gland, at 4 weeks post-induction with tamoxifen in adulthood and then immunostained for E-cadherin (
blue). Enlargements show optical sections depicting luminal cell-containing clones (
upper panel) or elongated myoepithelial cells (
lower panel).
Scale bars = 200 μm (whole-mounts) and 50 μm (enlargements)