To label slowly dividing SHZ cells a daily intraperitoneal Bromodeoxyuridine (BrdU; Sigma-Aldrich, 33.33 mg/kg) injections were given into CXCR4-EGFP mice, twice a day for 6 days, and mice were killed 24 h later after the last injection. To label rapidly dividing SHZ cells and their descendants, mice were injected once and killed 2 h later. Whole-mount fixed preparations or free floating fixed sections were then incubated in 1N HCl for 30 min at 45 °C and neutralized in PBS before immunostainings for BrdU (1/500, sheep, Fitzgerald Industries International, MA) and Ki67 (1/100, rabbit, BD Pharmingen, CA) for proliferating cells exiting the cell cycle.
Bromodeoxyuridine (brdu)
Manufactured by Biosynth
Sourced in United States
BrdU (5-Bromo-2'-deoxyuridine) is a synthetic thymidine analog that can be incorporated into DNA during cell division. It is commonly used as a marker for cell proliferation in various biological and biomedical applications.
Automatically generated - may contain errors
2 protocols using bromodeoxyuridine (brdu)
1
Labeling Slowly and Rapidly Dividing Cells
2
Histological Analysis of Mouse Uterus
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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 system41 (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).
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