Carmine Alum Wholemount Preparation

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.