Hrp conjugated rabbit anti mouse secondary antibody

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.