4.3.11.3 mouse mab

The experimental setup is shown in Supplementary Figure S1. Day 19 prepubertal C57BL/6J female mice were labeled with BrdU according to our previous study [18 (link)]. After a 6-week chase, the standard protocol to induce endometrial breakdown and repair was performed [19 (link)]. The BrdU⁺ cells after a 6-week chase were referred as LRSC in the present menstrual-like model. In brief, female mice were mated with vasectomized >6-week-old C57BL/6J male mice (day 0). Pseudopregnant female mice were identified by the presence of a vaginal plug on the next day (day 1). On day 4 of pseudopregnancy, 30 µL of sesame oil was injected into the left uterine horn to induce decidualization, while the right horn was not treated as control. The mice were euthanized, and their uteri were harvested on day 4 of pseudopregnancy (before decidualization), decidualization (day 7), breakdown (day 9), early repair (day 10) and late repair (day 12). Mice received an intraperitoneal injection of pimonidazole (60 mg/kg, 4.3.11.3 mouse MAb, Hypoxyprobe, Burlington, MA, USA) 1.5 h before being euthanized. The harvested uteri were fixed with 4% paraformaldehyde overnight and processed into paraffin blocks.

Five micrometer mouse uterine sections were stained with haematoxylin and eosin (H&E) and stage of breakdown/repair graded by two masked independent observers using a previously published scoring system^{41 (link)} (Supplementary Fig. 2). Mouse uterine sections were also stained for endoglin (1:50, R&D Systems, AF1320, Abingdon, UK), alpha smooth muscle actin (αSMA, 1:250, C6198, Sigma, Dorset, UK), Ly6G (BioLegend, 127601, 1:1000. London, UK), F4/80 (Bio-Rad, MCA497GA, 1:50. Oxford, UK) and carbonic anhydrase IX (Abcam, ab184006, 1:2000, Cambridge, UK). Proliferating cells were identified using anti-bromodeoxyuridine antibody (BrdU, 1:1500, Fitzgerald, Acton, MA, USA) and hypoxia detected using anti-pimonidazole antibody as per manufacturer’s instructions (4.3.11.3 mouse MAb, Hypoxyprobe, Burlington, USA).