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BT |
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Thiazol blue tetrazolium was added (10 [micro]L) and the plate was returned to thermostat for 5 h. The embryos were then blocked (1 hr) in 20% goat serum, incubated overnight in an antidigoxigenin antibody coupled to alkaline phosphatase (1:2,000) (Roche), washed extensively in PBS (at least 6 x 1 hr) and overnight in PBS, followed by a chromogenic nitro blue tetrazolium chloride (NBT) reaction [NBT/BCIP (5-bromo-4chloro-3-indolyl-phosphate, toluidine-salt) tablets, Roche] for 10-30 min, until color was visible. Sections were washed, and the color was developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) as the substrate and nitro blue tetrazolium salt (NBT, Sigma) as the chromogen for 1 hour at 37 [degrees] C. |
blue tetrazolium |
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