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

The following protocol was optimized from a protocol using 35S-labeled RNA probes [1, 2]. In research studies, it enables detection of the expression of rare mRNAs in the cardiovascular system (e.g., of the proinflammatory cytokine granulocyte macrophage colony stimulating factor [GM-CSF] in normal human coronary arteries, and of interleukin 6 [IL6] and glycoprotein gp130 in failing human hearts [3]). The protocol can be combined with immunohistochemistry [4, 5], and can also be used for paraffin- and methacrylate-embedded sections.

Materials and Methods

Preparation of DIG-labeled RNA probes

The desired cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the cDNA was cloned into an in vitro transcription vector. DIG-labeled RNA probes were transcribed in vitro from the plasmid according to the instructions of the DIG RNA Labeling Kit. Best results will be obtained with digoxigenin-labeled RNA probes of < 600 bp. For larger probes, adjusting the proteinase K digest is preferred to probe denaturation. The amount of DIG-labeling was determined by dot blot. It is important to determine the exact concentration of the RNA probes to avoid background after hybridization.

Tissue preparation

Tissue was frozen as soon as possible after excision to prevent degradation of mRNA. The tissue was cut to the appropriate size, and as much of the fatty tissue as possible was removed. The tissue was then immersed in a cryoprotective medium and frozen on cork disks in nitrogen- cooled 2-methylbutan. The tissue was stored at 80 C or in liquid nitrogen.

Preparation of cryosections
The tissue samples were prewarmed to 22 C and tissue sections were prepared (4 m12 m, thicker sections may be preferred for confocal microscopy). The sections (24) were placed on silanecoated slides and immediately used or stored at 80 C (up to several months).

Prehybridization procedure

The slides were dried for 1 hour at room temperature or in an oven for 10 minutes at 50 C. The sections were treated for 10 minutes with chloroform, if the tissue was rich in lipids. The sections were fixed for 10 minutes with phosphate-buffered 4% paraformaldehyde and rinsed 3 times in 5x TE (50 mM Tris-HCl pH 8.0, 5 mM EDTA). The sections were treated with proteinase K for 10 minutes at room temperature if necessary. Whether proteinase K treatment is required, and which concentration of proteinase K is used strongly depends on the kind of tissue and the fixation. For blood vessels and myocardial tissue we used:
  • Cryosections: up to 2 g/ml
  • Paraffin-embedded sections: up to 20 g/ml
  • Methacrylate-embedded sections: up to 50 g/ml
Sections were rinsed in Tris-Glycine (100 mM Tris-HCl pH7.0, 100 mM glycine), post-fixed for 10 minutes in 4% phosphate-buffered paraformaldehyde, and rinsed in TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) 3 times for 5 minutes. The sections were then rinsed in distilled water once (5 minutes), dehydrated in increasing concentrations of ethanol, and dried in a dust-free area. After the prehybridization procedure, the sections may be stored for a few days in a refrigerator. However, optimal results will be achieved by immediately continuing with in situ hybridization.

Hybridization procedure

Homologous probes were hybridized at 5052 C. For heterologous probes, we recommend lower temperatures. The hybridization solution (50% formamide, 2x SSPE buffer, 10mM DTT, 1mg/ml herring sperm DNA, 500 g/ml yeast tRNA, 1 mg/ml BSA) was denatured for 10 minutes at 80 C. The sections were preincubated in a humidified chamber for 2 hours in hybridization solution. The prehybridization solution was removed, and hybridization solution was added (concentration of the DIG-labeled RNA probe: 0.3 1 g/ml). The tightly sealed chambers were incubated overnight in a shaking water bath at 50 C.

Posthybridization procedure

The hybridization solution was removed by thoroughly rinsing the slides in 4x SSC. The slides were washed twice in 2x SSC for 15 minutes at 50 C and twice in 1x SSC for 15 minutes at 50 C. To remove nonspecifically bound DIG-labeled RNA probes, the slides were incubated with RNase A (10 g/ml NTE: 500 mM NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA) for 10 minutes at 37 C. The slides were then washed twice in 0.1x SSC for 10 minutes at 50 C.

Detection procedure

For detection, the DIG Nucleic Acid Detection Kit was used. The sections were washed for 5 minutes in buffer 1. Nonspecific background was blocked by incubating the sections for 1 hour in buffer 2 (buffer 1 with 0.5% Blocking Reagent).

  • Alkaline phosphatase conjugated antibodies

The sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Fab-fragments) (dilute 1:5001:1,000 in buffer 2) for 1 hour at room temperature, then rinsed thoroughly in buffer 1 containing 0.05% Tween, washed twice for 15 minutes, and incubated for another 15 minutes in buffer 3. The slides were then incubated with an appropriate amount of staining solution in a humidified chamber for 30 minutes to 24 hours (staining solution: 335 g NBT [stock solution: 75 mg/ml in 70 % dimethylformamide], 174 g BCIP [stock solution: 50 mg/ml in 100% dimethylformamide], and 240 g Levamisole per ml buffer 3). The staining solution was removed by rinsing in 5x TE, the staining procedure was stopped by incubating the slides for 15 minutes in 5x TE. The sections were briefly rinsed in distilled water, then counterstained with methylene green. The sections were mounted with Kaisers glycerin-gelatin. If immunofluorescence protocols are used in combination with in situ hybridization, we recommend development with the ELF substrate (ELF Kit, Molecular Probes).

  • Fluorochrome-conjugated antibodies

When the mRNA of interest is abundantly expressed, anti-DIG antibodies conjugated with FITC or other fluorochromes can be used. The slides were incubated for 2 hours with FITC-conjugated anti-DIG antibody (1:201:200 in buffer 1), then washed twice for 5 minutes with buffer 2 (Buffer 1 containing 0.05% Tween). The sections were counterstained with Hoechst Dye 33342 and mounted with fluorescence mounting medium (DAKO).

Immunohistochemistry

Usually immunohistochemistry was performed immediately after in situ hybridization. However, with both detection procedures excellent results were also obtained for several weeks (up to months) after in situ hybridization. All washing steps were performed on a shaking platform.

  • The peroxidase staining procedure was performed according to the manufacturers recommendations (Vectastain Elite Kit, Vector Laboratories).
  • Immunofluorescence: The slides were incubated with PBS/12% BSA for 12 hours in a humidified chamber, then the blocking solution was removed. The slides were incubated with an appropriate 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 muscle 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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