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Heparin @ 50 mg/ml (40C) Sigma H-3393 50μl.............................…….50μg/ml
20% Tween 250 μl...............................0.1%
0.5M Citric acid 920 μl..............................to …to pH 6
DEPC/water to 50 ml (store in --20°C)
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- Blocking step: incubate embryos in 1X WBR (dilute from the 10XWBR in PBT and filter it!!! Fisherbrand .22μm Cat# 09-719A) for 2 hours at RT.
5. Add anti-Dig-POD *(The reagent is an anti-digoxigenin antibody from sheep, Fab fragments, conjugated with polymerized horse-radish peroxidase [POD(p)]. (Roche* 11 633 716 001*)* final concentration of 1:100-1000 in filtered 1X WBR. Incubate on rocker overnight at 4°C.
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