BrDU protocol
Amount of injected BrDU is based on the calculation 50mg BrDU / 1kg weight. We usually inject
1.5 mg/mouse.
Dissolve BrDU (Sigma) in 7 mM NaOH/PBS to a final concentration of 16 mM (5 mg BrDU into 1
mL NaOH/PBS). Add the BrDU and shake, then heat to 37'C until in solution; cool before
injecting.
Perform an i.p. injection on pregnant females (~ 300 ul). Inject thymidine (25 mg) as a chaser
after 2 h.
After dissection, place tissues into a fresh fixative made with 30% 50 mM glycine and 70%
ethanol and fix for 1 h at room temperature (or PFA for 30 min or into a Methanol gradient for
about 1 h on ice). Wash samples once in PBS, treat with 2 M HCl for 30 min at room temperature,
wash again, and then process samples for immunocytochemistry.