Aluminium potassium sulphate

Excised mouse mammary glands were laid outstretched and flattened on a glass slide (SuperFrost^TM, Thermo Scientific). The slide with the mammary gland was then incubated in Canoy’s fixative (a solution of ethanol/chloroform (Merck)/acetic acid (Merck) in a 6:3:1 ratio, respectively) for 16 h at room temperature. The slide was then rehydrated by incubating in 70% ethanol, 15 min; 50% ethanol, 15 min; 20% ethanol, 15 min; and distilled water, 5 min. The tissue was then stained in Carmine red (a solution of 0.2% (w/v) Carmine red (Sigma-Aldrich, St. Louis, MO, USA) and 0.5% (w/v) aluminium potassium sulphate (Sigma-Aldrich), boiled for 20 min, filtered, with a crystal of thymol (Sigma-Aldrich) added as a preservative, then stored at 4 °C) for 16 h at room temperature. The tissue was then dehydrated by incubating in 70% ethanol, 15 min; 90% ethanol, 15 min; and 100% ethanol, 15 min and stored in histolene (Grale, Ringwood, VIC, Australia) for photography under a light microscope (Supplementary Figure S2).

Carmine alum whole mounts were carried out as previously described [16 (link), 17 (link)]. Briefly, fourth inguinal mammary glands were fixed in 10% neutral‐buffered formalin (NBF) (Leica) overnight at 4°C. Glands were dehydrated for 1 h in distilled water, then 1 h 70% ethanol and 1 h 100% ethanol before incubation in xylene overnight (VWR international). Rehydration was achieved by 1‐h incubation in 100% ethanol, 70% ethanol and distilled water, before staining with Carmine Alum solution at room temperature overnight (0·2% (w/v) carmine and 10 mM aluminium potassium sulphate (Sigma)). Tissue was dehydrated again and incubated overnight in xylene. Glands were then mounted with DPX (Leica), and 10× magnification stitched brightfield images were obtained using an EVOS FL auto2 microscope (Thermo Fisher). 5× brightfield images were obtained using the Zeiss Axio Imager M2 with Zen 2012 software. TEBs were counted as the average from at least 2 F.O.V. from each whole mount. All samples were blinded before measurements were taken.

Carmine alum wholemounts were prepared as described previously (Wilson et al., 2017 (link)). Briefly, fourth inguinal mammary glands were spread onto Superfrost Plus slides (VWR) and fixed overnight in 10% neutral buffered formalin (NBF) (Leica) at 4°C. Glands were dehydrated for 1 h in distilled water, followed by 70% ethanol and 100% ethanol before overnight incubation in xylene (VWR international). Tissue was rehydrated by a 1 h incubation in 100% ethanol, 70% ethanol and distilled water, before staining in carmine alum solution overnight at room temperature [0.2% (w/v) carmine and 10 mM aluminium potassium sulphate (Sigma)]. Tissue was dehydrated again before overnight incubation in xylene. Finally, glands were mounted with DPX (Leica) and stitched bright-field images at 10× magnification were taken using an EVOS FL auto2 microscope (ThermoFisher). Ductal elongation, and branched area from the lymph node, were measured using ImageJ 1.52a (Schneider et al., 2012 (link)). Bright-field images at 5× magnification were obtained using the Zeiss Axioimager M2 with Zen 2012 software. The numbers of branches and branch thickness were counted as the average from three measurements from six individual fields of view (FOV) from each wholemount. TEBs were counted as the average from at least two FOV from each wholemount. Sample identities were hidden before measurements were taken.