BrdU

5-Bromo-2′-deoxyuridine is a thymidine analog used as a mutagen in genetic research and as a marker of newly divided cells in neurogenesis research. Selectively incorporated into cellular DNA during S-phase.

Sigma Product Number: B5002

CAS Number: 59-14-3

Empirical Formula (Hill Notation): C9H11BrN2O5

Molecular Weight: 307.10

PubChem Substance ID:24278270

CAS Number: 59-14-3

Immunohistochemistry Protocol

Generously provided by Dr. Zuoxin Wang

BrdU Injections

50-300 μg/g body weight in 0.9% saline and 0.007N NaOH (injection volume of 0.1ml per vole).

Tissue Preparation

1. Deeply anesthetize animal, perfuse through the ascending aorta with saline (pH 7.4) followed by cold 4% paraformaldehyde in 0.1M PBS.
2. Postfix brain for 2-4 hrs at 4ºC, depending on the fixation.
3. Transfer brain into 30% sucrose in PBS until it sinks.
4. Cut brain on microtome and put sections into 0.1M PBS with 0.1% sodium azide and store at 4°C.

BrdU Immunocytochemistry

1. Rinse 3 X 5 min in 0.1M Phosphate Buffered Saline (PBS, pH7.4).
2. Incubate in 0.3% hydrogen peroxide and 10% methanol in 0.1M PBS for 15 min (use concentrated PBS to make solution).
3. Rinse 5 X 5 min in 0.1M PBS.
4. Incubate in 2N HCl in 0.1M PBS at 60°C for 30 min (use concentrated PBS to make solution).
5. Rinse 5 X 5 min in 0.1M PBS
6. Block in 0.5% Triton-X 100 and 10% Normal Goat Serum (NGS) in 0.1M PBS for 1 hr.
7. Incubate in 1:4,000 dilution of monoclonal rat anti-BrdU antibody (Accurate, OBT00300X) in 0.5% Triton-X 100 and 2% NGS in 0.1M PBS at 4°C overnight.
8. Incubate at room temperature for 1-2 hour.
9. Rinse 5 X 5 min in 0.5% TrintonX-100 in 0.1M PBS.
10. Incubate in 1:300 dilution of Biotin-conjugated goat anti-rat antibody in 0.5% Triton-X 100 and 2% NGS in 0.1M PBS for 2hr.
11. Rinse 5 X 5 min in 0.1M PBS.
12. ABC step: Vector Elite ABC Kit incubate for 90min (one drop A and one drop B in 5 ml 0.1M PBS, must be made at least 30 minutes before use to allow the formation of the avidin-biotin complex)
13. Rinse 5 X 5 min in 0.1M PBS.
14. Staining by DAB kit (Vector).
15. Rinse 5 X 5 min in 0.1M PBS.

Radiation Sensitization

Incorporation of BrdU makes cells more sensitive to radiation damage.