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Detection of mRNAs on Cryosections of the,,,Cardiovascular System Using DIG-Labeled RNA Probes

le cells, some smooth muscle cells of the tunica intima (Figure 1A), about 50 % of the endothelial cells (Figure 1B), and only a few macrophages located in the tunica adventitia (Figure 1C). In all stages of plaque development, GM-CSF was coexpressed with type VIII collagen [5] (Figure 2). Using the protocol described above, we easily characterized the GM-CSF and type VIII collagen-expressing cell types in cryosections and paraffin-embedded samples of various arteries, in the myocardium, and in cultured vascular cells.
As shown in Figures 1 and 2, the protocol described represents an excellent, easy to use means to evaluate the spatial and temporal expression pattern of mRNAs in various tissues and cultured cells. The protocol allows one to simultaneously characterize the expressing cell types, as well as the localization and distribution of other proteins. The protocol is as sensitive as radioactive in situ hybridization. However, it is less time consuming [1, 2] and more precisely locates the expressing cells.

One critical factor for converting the protocol from radiolabeled probes to nonradioactively-labeled probes has been the use of the highly sensitive DIG-labeled cRNA probes. Together with the enhancement of sensitivity by the DIG system, the use of the related products such as the DIG RNA Labeling Kit or the DIG Nucleic Acid Detection Kit provides in situ hybridization of consistently excellent quality.


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