Antidigoxigenin FAb

This chapter describes a nonradioactive method for the localization of mRNA in whole mouse embryos. It employs riboprobes labeled with digoxigenin, a steroid-like moiety not found in animal tissue. Digoxigenin-containing probe is visualized with a conjugate of antidigoxigenin Fab and alkaline phosphatase and colorimetric staining. The results are visualized in three dimensions, hence subtle patterns can be visualized without laborious sectioning. 

At this point begin preadsorbing the antidigoxigenin Fab/alkaline phosphatase conjugate see Section 2.). 

Wipe the slide dry around the tissue but keep the tissue area wet. Put onto the each section 200 pL of antidigoxigenin antibody (a Fab fragment), diluted 1 200 in buffer 1. Incubate at least for 2 h at room temperature. 

Rhodamine-conjugated antidigoxigenin sheep IgG, Fab fragments (Boehringer Mannheim 2 pg/ml, diluted in SBT)... 

To each slide, apply 80 xl of 2 Xg/ml rhodamine-conjugated antidigoxigenin sheep IgG, Fab fragments, diluted in SET. Cover with a 22 X 22-mm coverslip and incubate at 37°C for 30 minutes in a dark, humid chamber. 

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