Brdu staining kit

DNA synthesis of cells was assayed by bromodeoxyuridine (BrdU). Pregnant females were intraperitoneally injected with BrdU 100 mg/kg. A BrdU Staining Kit (Invitrogen) was used for analysis. Detection of apoptosis was performed by TUNEL staining using the In Situ Apoptosis Detection Kit (Takara Bio) according the manufacture's protocol.

Six-day-old Twist1^flox/+; Twist2^cre/+ mice and control mice were intraperitoneally injected with 5-bromo-2′-deoxyuridine (BrdU) (10 mg/100 g body weight) at 24 hours and then two hours before sacrifice. The long bones were collected and processed for paraffin sectioning as described above. The incorporated BrdU was detected with a BrdU staining kit (Invitrogen, Camarillo, CA, USA) according to the manufacturer's instructions. The BrdU-positive nuclei and total nuclei were counted in the metaphysis region (trabecular bone) as well as in the midshaft (cortical bone). The data represented the percentage of BrdU-positive nuclei from four individual animals each group.

Eight-week-old Dgcr8^f/f and Dgcr8^d/d mice were ovariectomized, rested for 2 weeks, and treated subcutaneously with either 0.1 ml sesame oil as vehicle (Acros, NJ, USA), 200 ng E₂ (Sigma-Aldrich), or 200 ng E₂ + 2 mg P₄ (Sigma-Aldrich). Mice were sacrificed 24 h after steroid hormone treatment(s). 5-Bromo-2´-Deoxyuridine (BrdU) (Invitrogen Life Technologies) was given to the mice 3 h before they were sacrificed. Uteri were dissected, fixed in 4% paraformaldehyde and then subjected to paraffin-embedded tissue processing. Uterine sections were utilized for BrdU immunostaining using a BrdU staining kit (Invitrogen Life Technologies), according to the manufacturer’s instructions.
Uterine apoptosis of Dgcr8^d/d mice was assessed using an In Situ Cell Death Detection Kit according to the manufacturer instructions (Roche, West Sussex, UK). Sections were deparaffinized and rehydrated in a graded alcohol series, and then processed for antigen retrieval. They were incubated with TUNEL reaction mixture for 1 h at 37 °C and then with DAPI for 10 min at room temperature, and observed under a fluorescence microscope.

Skeletal preparations were stained with alizarin red S and alcian blue, as described previously [4 (link)]. For light microscopy, we fixed tissues from embryos at E14.5, E16.5, and E18.5 in 4% paraformaldehyde/0.1 M phosphate buffer, embedded them in paraffin, and prepared 7-μm sections to perform H-E staining, safranin O staining, and TRAP staining. For the analysis of cell proliferation, we intra-peritoneally injected pregnant mice with 50 μg of BrdU/g of body weight 1 h before sacrifice. We processed the embryos for histological analysis and detected BrdU incorporation using a BrdU staining kit (Invitrogen, Carlsbad, CA, USA). Apoptosis was analyzed by TUNEL staining using the ApopTag peroxidase In Situ apoptosis detection kit (Sigma-Aldrich, St. Louis, MO, USA). The frequencies of BrdU- or TUNEL-positive cells were calculated using ImageJ software.

Mouse BMMSCs (10 × 10³/well) were seeded on 2-well chamber slides (Nunc, Rochester, NY, USA) and cultured for 2–3 days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 h, and stained with a BrdU staining kit (Invitrogen) according to the manufacturer's instructions. The samples were then stained with hematoxylin. BrdU-positive and total cell numbers were counted in 10 images per subject. The number of BrdU-positive cells was indicated as a percentage of the total cell number. The BrdU assay was repeated on three independent samples for each experimental group.

Cells were plated in chamber slides (30,000 cells/well) and serum starved for 48 hours. Next, cell culture medium supplemented with 10% FBS was added for 24 hours followed by bromodeoxyuridine reagent incubation for 2 hours at 37°C. Bromodeoxyuridine (BrdU) incorporation in proliferating cells was estimated using an immunohistochemical analysis kit (Invitrogen, BrdU Staining Kit). The BrdU-labeling index, expressed as the percentage of cells labeled with BrdU, was determined by counting 1000 cells in 3 independent reactions using the Kontron 400 image analysis system (Zeiss Axio Imager A1, Dublin, California, USA).