Carmine Alum Whole Mount Imaging

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.