5 bromo 2 deoxyuridine brdu

Adult male C57BL/6 mice (weighing 22–28 g, aged 8–12 weeks) were obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All animal handling procedures were performed in accordance with the Chinese guidelines for animal welfare. The animal study protocol was approved by the Animal Ethics Committee of Henan University of Science and Technology.
Recombinant mouse GDF-5 was obtained from R&D Systems (Minneapolis, MN, USA). 5-bromo-2′-deoxyuridine (BrdU) was from Thermo Fisher Scientific (Waltham, MA, USA). The antibody against BrdU was purchased from Abcam (Cambridge, UK; AB6326). The antibodies against doublecortin (DCX) and Sox-2 were from Santa Cruz Biotechnology (Dallas, TX, USA; sc-8066 and sc-17320). The antibodies against phosphorylated cAMP response element binding protein (p-CREB, Ser 133), NeuN, and c-Fos were from Millipore (Billerica, MA, USA; 06-519, MAB377, and PC05).

After 5 days of culturing, BM-EPCs were labeled with 5-bromo-2′-deoxyuridine (BrdU; Thermo Fisher Scientific; Waltham, MA, United States) as previously described (Peng et al., 2018 (link)). In brief, endothelial growth medium-2 (EGM-2; Cambrex Corp; East Rutherford, NJ, United States) was used to dilute BrdU-labeled reagent (1:100), and the mixture was filtered with a 0.2 μm filter and heated to 37°C. A total of 2 ml of EGM-2 containing BrdU was added to cells that were plated in a six-well plate. On day 7, cells were washed with PBS for three times and harvested with 0.125% trypsin. Sprague-Dawley rat EPCs (400,000 cells) were transplanted into VaD rat via the tail vein. After 7 days of a single injection of EPCs, rats were sacrificed, and brain tissues were collected for immunofluorescence staining.

KSHV lytic cycle was induced in BCBL-1 cells using the 12-O-tetradecanoyl phorbol-13-acetate (TPA; 20 ng/ml). Virion productions and purifications were carried out as per our methods described previously (Roy et al., 2016 (link)). To quantify the copy number of the virions, KSHV DNA was extracted and quantified by real-time DNA-PCR using primers specific for the KSHV ORF73 gene as described previously (Roy et al., 2016 (link)). TREX-BCBL-1-RTA cells were induced with doxycycline (DOX, 1 µg/ml).
For de novo KSHV infection, TIME or U2OS cells were washed twice with phosphate buffer saline (PBS), infected with 100 genome copies/cell in serum-free basal medium for 2 hr, washed with PBS and incubated in complete medium from 24 to 96 hr, as indicated.
For the production of BrdU and EdU labeled KSHV, viral DNA was labeled by adding 5-Bromo-2'-deoxyuridine (BrdU) (Thermo Scientific # B23151) 1:100 v/v or 5-ethynyl-2'-deoxyuridine (EdU) (Thermo Scientific #A10044) 10 µM in DMSO into the culture medium of TPA induced BCBL-1 cells. The viral DNA is metabolically labeled during lytic replication. BrdU/EdU was added twice in the culture medium, once on day 1 of TPA induction and again on day 3. The labeled virus from day 5 culture was purified and genome copy number determined as described earlier.

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.