A. Virgin mammary glands (30 and 70 days old) showed no conspicuous basal membrane, as visualised by collagen IV IHC (a, b). There was also no detectable expression of HIF-2α in the epithelial cells (c, d). Macrophages (F4/80 positive) were few. In panel c, a single HIF-2α-positive cell was detected, and the adjacent F4/80 IHC section (e) suggested that this cell is a macrophage. B. Expression of HIF-1α in mammary epithelium in the 70-day-old virgin mouse. Top panel, orientation slide with haematoxylin (HTX) staining, 20x obj. *lymph node. Panels b, d, f. Cross-section of a developing duct close to the invading tip at a stage where the lumen is not yet evacuated, 40x obj. Panels c, e, g. Cross-section of a less mature part of a duct, 40x obj. CK14-expressing cells (marker of basal mammary epithelial cells) can be seen in more than one cell layer (panels b and c, arrow-head). At this stage, the lumen is evacuated, but there is still more than one layer of epithelial cells. HIF-1α IHC on the adjacent sections (panels d, e) showing nuclear expression in non-basal epithelial cells (highlighted by red arrows). Basal (CK14 positive) epithelial cells did not express HIF-1α (black arrows). Mammary epithelial expression of HIF-2α was not detected at these developmental stages. Size bars: 50 μm.