Methods for double detection of gene expression. Combined in situ hybridization and immunocytochemistry of histochemistry.

The distribution of two different molecules can be analyzed within the same embryo using the procedures described below. The protocol for combined whole mount in situ hybridization and immunocytochemistry allows for simultaneous detection of mRNA and protein. The protocol for combined whole mount in situ hybridization and β-galactosidase staining allows for simultaneous detection of mRNA and transgene-directed β-galactosidase expression. Simultaneous detection allows for the most direct comparison of expression patterns. These procedures are derived from protocols used in Drosophila (1) and mice (2, 3). The required materials include those for whole mount in situ hybridization, plus the following reagents. 3. Primary antibody against the protein of interest. 4. An appropriate secondary antibody conjugated to horse radish peroxidase. 5. Anti-digoxigenin antibody, alkaline phosphatase-conjugated (Boehringer Mannheim). Store at 4°C. 6. Blocking Reagent for nucleic acid hybridization (Boehringer Mannheim). 7. DAB (30 mg/ml 3, 3'-diaminobenzidine tetrahydrochloride in 10 mM Tris pH 7.6 stored at-20°C in the dark in single use aliquots). Handle with extreme caution: DAB is carcinogenic. 8. BCIP (50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt in 100% N,N-dimethylformamide, stored at-20°C in the dark).