DOUBLE LABELING WITH NONISOTOPIC INSITU HYBRIDIZATION AND BRDU IMMUNOHISTOCHEMISTRY - CALMODULIN (CAM) MESSENGER-RNA EXPRESSION IN POSTMITOTIC NEURONS OF THE OLFACTORY SYSTEM

We describe a novel histochemical procedure for simultaneous detection of mRNA expression by in situ hybridization (ISH) and DNA synthesis o n cells that are pulse-labeled with bromodeoxyuridine (BrdU) by immuno histochemistry (ICC). Pregnant rats were injected with BrdU at embryon ic Day 20 and the olfactory bulbs of their pups were collected daily. The expression of calmodulin (CaM) mRNA was analyzed by ISH with an an ti-sense digoxigenin-labeled riboprobe and BrdU incorporation by indir ect ICC. Starting 5 days after BrdU injection, a few tufted and granul ar neurons of the olfactory bulb were observed to be double labeled fo r CaM mRNA and BrdU. To study the olfactory neuroepithelium, adult ani mals were injected with BrdU, sacrificed after 30 days, and the nasal mucosa dissected and decalcified. The co-expression of CaM mRNA and Br dU incorporation was then analyzed in the olfactory neuroepithelium: B rdU-positive primary olfactory neurons were also CaM mRNA positive. Th e combination of ISH and ICC on the same section resulted in improved BrdU staining with respect to both increased intensity and reduced bac kground levels. The procedure described here can be applied to a varie ty of problems in developmental biology and is of potential value for correlating the timing of specific mRNA expression with the birth date of a cell type of interest.