Fluorescent BrdU assay kit

In vitro labeling

• Dilute BrdU stock 1:1,000 with sterile cell culture medium.
• Incubate the cells at 37°C and 5% CO₂ for 10 to 60 min.
• Aspirate the BrdU labeling solution.
• Wash cells with 1xPBS three times.
• Fix cells with cold methanol for 30 min at −20°C.
• Aspirate methanol and let cells air-dry for 30 min.
• Add antibody Block solution to fixed cells and incubate at room temperature for 30min.
• Aspirate antibody Block solution.
• Dilute anti-BrdU primary antibody at 1:50 in antibody Block solution and add to fixed cells.
• Incubate the cells at room temperature for 2hr.
• Aspirate antibody labeling solution and wash fixed cells with antibody Wash solution three times.
• Dilute FITC conjugated secondary antibodies at 1: 200 in antibody Block solution and add to fixed cells.
• Incubate the cells at room temperature for 1hr.
• Aspirate antibody labeling solution and wash fixed cells with antibody Wash solution three times.
• Add 20 ul of Mowiol solution to the fixed cells if using a chamber slide. Place a cover glass onto Mowiol solution and apply a small pressure to remove any trapped air bubble. Let Mowiol solution dry at room temperature for 1hr. Store chamber slides at 4^oC for less than 1 month.

In vivo labeling

• Dilute BrdU stock 1:10 with 1xPBS.
• Inject animal intravenously with diluted BrdU at 1 ml/10 g body weight.
• Sacrifice animal 1 hour after injection and remove selected organs.
• Process tissue for frozen sectioning or formalin fixing and paraffin-embedding.
• Deparaffinize FFPE sections with Xylene.
• Rehydrate sections with decreasing concentrations of ethyl alcohol (100% to 70%).
• Wash slides 2 times with dH₂O.
• Dip slides into universal antigen retrieval buffer.
• Microwave sections at boiling temperature for 10min.
• Wash slides 2 times with dH₂O.
• Wash slides with 1xPBS.
• Drain PBS against a towel and put slides into the wet chamber.
• Dilute anti-BrdU primary antibody at 1:50 in antibody Block solution and add to each slide (300-500 µl is sufficient to cover the section on each slide).
• Incubate the slides in the wet chamber at room temperature for 2hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the antibody Wash solution.
• Dilute FITC conjugated secondary antibodies at 1: 200 in antibody Block solution and add to each slide and put slides back into the wet chamber.
• Incubate the slides in the wet chamber at room temperature for 1hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the antibody Wash solution.
• Add 20 ul of Mowiol solution to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble. Let Mowiol solution dry at room temperature for 1hr. Store glass slides at 4^oC for less than 1 month.