As an alternative to sequential AP-conjugated antibodies, one can use antifluorescein-Fab fragments conjugated to AP and Vector Red to identify the first signal followed by antidigoxigenin Fab-conjugated to horseradish peroxidase. The second staining reaction is performed with TrueBlue™ (Kirkegaard Perry). TrueBlue™ is an alternative peroxidase substrate to diaminobenzidine (DAB). It is 50 times more sensitive than DAB, so the antidigoxigenin Fab-conjugated to horseradish peroxidase must be used at 10- to 50-fold greater dilution. This method overcomes the problem of possible residual AP activity giving rise to a false-positive colocalized signal. However, the TrueBlue™ precipitate is not stable in aqueous solution, so specimens must be photographed immediately or rapidly dehydrated and transferred to organic mountant.
Proceed as in Subheading 3.3.6. up to step 5, and then as follows. Note that the glycine inactivation step is omitted.
1. Incubate in Vector Red™ staining solution for a few hours to overnight. When color has developed to the desired extent, wash 3X with PBT.
2. Replace PBT with blocking solution, and incubate for a further 1 h.
3. Incubate for at least 2 h with sheep antidigoxigenin-horseradish peroxidase Fab fragments at a concentration of 0.015-0.0375 U/mL.
5. Transfer to a embryo dish, and add TrueBlue™ staining solution.
6. Monitor staining and photograph.
7. If overstained, replace staining solution with PBT to destain.
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