Anti brdu monoclonal antibody

Before ending the experiment (30 min prior to sacrificing the mice), the tumor bearing animals were injected i.p. with 5-bromo-2 V-deoxyuridine (BrdU, 200 mg/kg; Sigma-Aldrich Kft, Budapest, Hungary). After 6 h, 5–7 μm thick tissue sections were excised from the newly frozen tumors and used for the detection of BrdU positive cells using an anti-BrdU monoclonal antibody (Becton Dickinson Hungary Kft., Budapest, Hungary), according to the manufacturer’s protocol. Positive cells were visualized with TRITC-conjugated anti-mouse IgG (1:100, Sigma). Endothelial cells were distinguished from tumor cells by treating the samples with rat anti-mouse CD31 antibody and labelled with biotinylated anti-rat IgG and streptavidin-FITC (Vector Laboratories, Burlingame, CA). The nuclei were stained with the Hoechst 33,342 dye (Molecular Probes, Eugene, OR). The number of labelled HT-29 tumor cells and the quantity of CD31-positive samples were determined in two different tumors by microscopic analysis of 6 independent regions. The vascularization extent of primary tumors was determined by microscopic analysis of two different living tumors, where four independent areas of living cells were counted and the percentage of separated endothelial cells covering a 10× magnification field of vision was calculated [12 (link)].

BrdU (Sigma) was added to neuronal cultures at a final concentration of 10 µM. The cells were incubated for 4 h and then fixed in 10% formaldehyde for 10 min at room temperature. The DNA was denatured by incubation with 2 N HCl for 30 min at room temperature, followed by neutralization with 0.1 M borate buffer, pH 8.5. The incorporation of BrdU into the nucleus was assayed by immunofluorescence using an anti-BrdU monoclonal antibody (1∶100; Becton Dickinson, Franklin Lakes, NJ), followed by counterstaining of the cells with Hoechst 33258. The number of BrdU-positive nuclei were divided by the total number of Hoechst-stained nuclei and expressed as a percentage of the total number of nuclei.

MDA-MB-231 cells were incubated with BrdU and uridine for 48 h. Then BrdU labeling assay was performed as described before [50 (link)]. Briefly, cultures were incubated with 1 mg ml⁻¹ (3 mM) BrdU and 1 mg ml⁻¹ uridine for 48 h. The cells were washed with PBS, fixed with ice-cold absolute methanol for 10 min, treated with 1.5 M HCl for 1 h at room temperature, and neutralized with 0.1 M borate buffer (pH 8.5) for 30 min. After blocking with 0.1% bovine serum albumin in PBS for 30 min at 37 °C, the cells were incubated with 5 mg ml⁻¹ anti-BrdU monoclonal antibody (Pharmingen, Franklin Lakes, NJ, USA) in 0.1% PBS/bovine serum albumin for 1 h, washed with PBS/bovine serum albumin and incubated with 1 mg ml⁻¹ HRP-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. The cells were then washed extensively with ammonia-buffered phosphate (0.1 M NaH₂PO₄ brought to pH 7.0 with ammonium hydroxide) and stained for 12–16 h at room temperature with 1.3 mM 3,39-diaminobenzidine in ammonia-buffered phosphate containing 0.004% H₂O₂.