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 ). 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 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).
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.
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.
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.
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%
- 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).
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).
Results and Discussion
- 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).
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 . 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  (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
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