28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
351
images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the target analyte to
be localized and evaluated.
Digital images are stored in the computer and quantitatively analyzed. The
light emission from each cell can be quantified by defining a fixed area and
summing the total number of photon fluxes from within this area. The
light emission is then expressed as photons/second/area (Fig. 1).
- Sections (3-5 11m thin) of tissue samples, either fixed in formalin and Samples
embedded in paraffin or frozen in liquid nitrogen and stored at -80°C
until use, are cut and placed on pretreated or silanated slides. Paraffin
embedded tissue sections are dewaxed by two 5-min incubations in
xylene and then washed in absolute ethanol. Frozen sections are fixed
with 4% paraformaldehyde in PBS for 30 min. After fixation, the slides
are washed three times in PBS for 10 min each and then the samples are
dehydrated with ethanol washes (30%,60%, 80%, 95% and 100%) for
5 min each. The slides are air dried and stored at 4°C. Samples can
be stored for several months with no decrease in their interaction
with the hybridization probes.
- Cellular specimens from body fluids are centrifuged at 1000 rpm for 10
min at room temperature, resuspended in 0.15 M PBS, pH 7.4, and
smeared or cytospun onto pretreated or silanated glass slides. For
blood-nucleated cells, a previous separation from peripheral blood
by Ficoll gradient is recommended. Cellular samples are fixed and dehydrated as described above for frozen sections.
The probes used for chemiluminescence in situ hybridization (ISH) gen- Probes
erally consist of double stranded DNA probes obtained from cloned DNA
fragments but they can also consist of synthetic oligonucleotides, single
stranded DNA probes generated by PCR, or single-stranded antisense
RNA probes. Synthetic oligonucleotide probes, because of their small
size, have good penetration properties, and the fact that they are single-stranded, rules out the possibility of annealing with complementary
strands in the hybridization solution. Cloned probes, which are usually
longer, have the advantage of having more sites available for labeling
and of recognizing more of the target DNA, resulting in a stronger signal.
The probes used in chemiluminescence in situ hybridization are nonisotopically labeled probes, with indirect labels such as biotin, fluorescein
or digoxigenin. These indirect labels are generally revealed by streptavidin, anti-fluorescein antibody and anti-digoxigenin antibody, respec-
351
images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the target analyte to
be localized and evaluated.
Digital images are stored in the computer and quantitatively analyzed. The
light emission from each cell can be quantified by defining a fixed area and
summing the total number of photon fluxes from within this area. The
light emission is then expressed as photons/second/area (Fig. 1).
- Sections (3-5 11m thin) of tissue samples, either fixed in formalin and Samples
embedded in paraffin or frozen in liquid nitrogen and stored at -80°C
until use, are cut and placed on pretreated or silanated slides. Paraffin
embedded tissue sections are dewaxed by two 5-min incubations in
xylene and then washed in absolute ethanol. Frozen sections are fixed
with 4% paraformaldehyde in PBS for 30 min. After fixation, the slides
are washed three times in PBS for 10 min each and then the samples are
dehydrated with ethanol washes (30%,60%, 80%, 95% and 100%) for
5 min each. The slides are air dried and stored at 4°C. Samples can
be stored for several months with no decrease in their interaction
with the hybridization probes.
- Cellular specimens from body fluids are centrifuged at 1000 rpm for 10
min at room temperature, resuspended in 0.15 M PBS, pH 7.4, and
smeared or cytospun onto pretreated or silanated glass slides. For
blood-nucleated cells, a previous separation from peripheral blood
by Ficoll gradient is recommended. Cellular samples are fixed and dehydrated as described above for frozen sections.
The probes used for chemiluminescence in situ hybridization (ISH) gen- Probes
erally consist of double stranded DNA probes obtained from cloned DNA
fragments but they can also consist of synthetic oligonucleotides, single
stranded DNA probes generated by PCR, or single-stranded antisense
RNA probes. Synthetic oligonucleotide probes, because of their small
size, have good penetration properties, and the fact that they are single-stranded, rules out the possibility of annealing with complementary
strands in the hybridization solution. Cloned probes, which are usually
longer, have the advantage of having more sites available for labeling
and of recognizing more of the target DNA, resulting in a stronger signal.
The probes used in chemiluminescence in situ hybridization are nonisotopically labeled probes, with indirect labels such as biotin, fluorescein
or digoxigenin. These indirect labels are generally revealed by streptavidin, anti-fluorescein antibody and anti-digoxigenin antibody, respec-