Bromodeoxyuridine (BrdU) Incorporation
(RL Erickson, 5/01)
Reagents/Solutions:
Stock BrdU: 10mM BrdU in sterile 10mM phosphate buffered saline. (I actually used the premade stock from the Roche BrdU kit but you can only buy the crystals individually.)
Antibody: FITC-conjugated Mouse Anti-BrdU Antibody Set (BD PharMingen Catalog No. 556028; comes with isotype control).
Fixitive: 70% Ethanol (ice-cold)
Wash Buffer: PBS with 0.5% BSA
Denaturing Sol'n: 2M HCl (MAKE FRESH)
Neutralizing Buffer: 0.1M Sodium Borate, pH 8.5
Dilution Buffer: PBS with 0.5% Tween-20 and 0.5% BSA
Protocol:
Plate 3T3-L1 preadipocytes in 6-well plates containing glass coverslips.
Two days post-confluence, induce the cells with MDI (see adipocyte differentiation protocol).
12 hours post-MDI add BrdU to media to a final concentration of 10 µM (1:1000).
24 hours post-MDI aspirate media and wash once with 2 mL of wash buffer.
Slowly add 1 mL of ice-cold 70% ethanol. Incubate for 20 minutes at RT.
Aspirate ethanol and wash once with 2 mL of wash buffer. Allow wash buffer to sit for 5 minutes. Aspirate wash buffer.
Add 2 mL of denaturing solution to cells. Incubate for 20 minutes at RT.
Aspirate denaturing solution and add 2 mL of wash buffer for 5 minutes. Aspirate wash buffer.
Add 2 mL of neutralizing buffer. Incubate for 2 minutes at RT
Aspirate neutralizing buffer and add 2 mL of wash buffer.
(THE REMAINING STEPS SHOULD BE PERFORMED IN REDUCED LIGHTING)
Prepare antibody solution (70uL per sample) by diluting 20 µL of anti-BrdU-FITC antibody in 50 µL of dilution buffer.
Aspirate wash buffer well, ensuring that the cover slip has no buffer surrounding it.
Apply antibody solution dropwise on to the coverslip. The surface tension should keep the solution on the slide. Incubate for 30 minute IN THE DARK at RT.
Wash coverslips twice with 2 mL of wash buffer.
Lift and prop coverslips against the side of the well to allow them to become semi-dry.
Mount on microscope slides using ProLong Antifade Kit from Molecular Probes.