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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.
[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=B5002|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma Product Number: B5002]
CAS Number: 59-14-3
Empirical Formula (Hill Notation): C9H11BrN2O5
Molecular Weight: 307.10
[http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=24278270 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==
#Deeply anesthetize animal, perfuse through the ascending aorta with saline (pH 7.4) followed by cold 4% paraformaldehyde in 0.1M PBS.
#Postfix brain for 2-4 hrs at 4ºC, depending on the fixation.
#Transfer brain into 30% sucrose in PBS until it sinks.
#Cut brain on microtome and put sections into 0.1M PBS with 0.1% sodium azide and store at 4°C.
==BrdU Immunocytochemistry==
#Rinse 3 X 5 min in 0.1M Phosphate Buffered Saline (PBS, pH7.4).
#Incubate in 0.3% hydrogen peroxide and 10% methanol in 0.1M PBS for 15 min (use concentrated PBS to make solution).
#Rinse 5 X 5 min in 0.1M PBS.
#Incubate in 2N HCl in 0.1M PBS at 60°C for 30 min (use concentrated PBS to make solution).
#Rinse 5 X 5 min in 0.1M PBS
#Block in 0.5% Triton-X 100 and 10% Normal Goat Serum (NGS) in 0.1M PBS for 1 hr.
#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.
#Incubate at room temperature for 1-2 hour.
#Rinse 5 X 5 min in 0.5% TrintonX-100 in 0.1M PBS.
#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.
#Rinse 5 X 5 min in 0.1M PBS.
#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)
#Rinse 5 X 5 min in 0.1M PBS.
#Staining by DAB kit (Vector).
#Rinse 5 X 5 min in 0.1M PBS.
=Radiation Sensitization=
Incorporation of BrdU makes cells more sensitive to radiation damage.
[[Category:Flavor Preference]]
[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=B5002|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma Product Number: B5002]
CAS Number: 59-14-3
Empirical Formula (Hill Notation): C9H11BrN2O5
Molecular Weight: 307.10
[http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=24278270 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==
#Deeply anesthetize animal, perfuse through the ascending aorta with saline (pH 7.4) followed by cold 4% paraformaldehyde in 0.1M PBS.
#Postfix brain for 2-4 hrs at 4ºC, depending on the fixation.
#Transfer brain into 30% sucrose in PBS until it sinks.
#Cut brain on microtome and put sections into 0.1M PBS with 0.1% sodium azide and store at 4°C.
==BrdU Immunocytochemistry==
#Rinse 3 X 5 min in 0.1M Phosphate Buffered Saline (PBS, pH7.4).
#Incubate in 0.3% hydrogen peroxide and 10% methanol in 0.1M PBS for 15 min (use concentrated PBS to make solution).
#Rinse 5 X 5 min in 0.1M PBS.
#Incubate in 2N HCl in 0.1M PBS at 60°C for 30 min (use concentrated PBS to make solution).
#Rinse 5 X 5 min in 0.1M PBS
#Block in 0.5% Triton-X 100 and 10% Normal Goat Serum (NGS) in 0.1M PBS for 1 hr.
#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.
#Incubate at room temperature for 1-2 hour.
#Rinse 5 X 5 min in 0.5% TrintonX-100 in 0.1M PBS.
#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.
#Rinse 5 X 5 min in 0.1M PBS.
#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)
#Rinse 5 X 5 min in 0.1M PBS.
#Staining by DAB kit (Vector).
#Rinse 5 X 5 min in 0.1M PBS.
=Radiation Sensitization=
Incorporation of BrdU makes cells more sensitive to radiation damage.
[[Category:Flavor Preference]]