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

opriate dilution of the respective primary antibody (in PBS /12% BSA) for 14 hours at room temperature or overnight at 4 C in a humidified chamber. The excess antibody was removed by washing three-times for 5 minutes in PBS that contained 0.05% Tween. The slides were then incubated with an appropriate dilution (1:5001:1000 in PBS /12% BSA) of the secondary antibody (for immunoconfocal detection, we recommend Cy-conjugated antibodies such as provided by Chemicon), then washed three times for 5 minutes in PBS, counterstained with Hoechst Dye 33342, and mounted with fluorescence mounting medium (DAKO).
Results and Discussion

Combined in situ hybridization and immunohistochemical staining has been used to identify vascular cells expressing GM-CSF and type VIII collagen in human coronary arteries. This non-radioactive procedure combined
  • in situ hybridization with DIG-labeled cRNA probes (GM-CSF, type VIII collagen) and
  • immunohistochemical characterization of vascular cells using cell type-specific antibodies (smooth muscle cells: anti smooth muscle actin, Enzo; endothelial cells: von Willebrand factor, Sigma; macrophages: CD68, DAKO) and a peroxidase staining procedure (Vectastain Elite Kit, Vector Laboratories). Approximately 70 % of the antibodies tested worked in this protocol.
The method enabled us to identify the intimal and medial smooth muscle cells as the major cell type expressing granulocyte macrophage colony stimulating factor (GMCSF) in the development of atherosclerotic plaques, particularly in advanced lesions [4]. Other GM-CSF-expressing cell types found in advanced lesions are endothelial cells and macrophages. In early lesions, GM-CSF mRNA (in situ hybridization, purple stain) was expressed mainly by medial smooth musc
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