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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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- Make TWS (tyramide working solution) just prior to the signal detection. To make TWS dilute the tyramide stock 1:100-1000 in 0.0015% H2O2/ amplification buffer (AB). Need about 200m200μl of TWS /well. Do serial dilution to make 0.0015% H2O2/AB: first make 0.15% H2O2 in AB (1:200 from 30% stock H2O2), then make AB/0.0015% H2O2 (1:100 from the 0.15% stock H2O2).
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4. Can look at embryos on an epifluorescence or confocal microscope at this point or continue with detection of next hapten. Wash longer if high background.
Second color reaction:
Begin protocol for 10 Antibody labeling for the 2nd probe:
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2) Make DMF-TEA solution:
5ml DMF
50ul 50μl TEA
3) Make reactive tyramine solution:
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