5-Bromo-2′-deoxyuridine incorporation and immunohistochemistry

Twenty-week-old mice were intraperitoneally injected with 5-bromo-2′-deoxyuridine (BrdU; 3 mg/ml in 100 µl ddH₂O) 2 h prior to euthanasia. Collected tissues from the right inguinal #4 mammary glands were prepared by FFPE for immunohistochemistry (IHC) analysis according to standard procedures. FFPE sections of collected mammary glands were deparaffinized with xylene, followed by rehydration with ethanol. Next, antigen retrieval was performed by boiling the tissue sections in citrate buffer for 30 min. Endogenous peroxidase and unspecific binding were then blocked with incubation in 3% hydrogen peroxide for 10 min, followed by DNA denaturation with 2N HCl for 30 min at 37°C. After incubation in 20% horse serum, the tissue sections were incubated in the BrdU or ERα primary antibodies overnight at 4°C while rocking. The next day, the tissue sections were washed with phosphate-buffered saline with Tween 20 and incubated in appropriate horseradish peroxidase (HRP) secondary antibodies for 1 h at room temperature. The tissues were then stained using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine. The sections were counterstained with hematoxylin, dehydrated and cleared. Finally, the stained sections were mounted and imaged using a Nikon Eclipse 80i microscope and NIS-Elements Microscope Imaging Software. The number of MECs with BrdU staining was recorded to determine the percentage of proliferating cells in each sample.