28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
355
No luminescent signal should be detectable in target positive cells/sections after hybridization with unlabelled probes, followed by the immunoenzymatic chemiluminescent treatment.
Target positive cells/sections must be completely negative after hybridization with the labeled probes when the primary incubation with antidigoxigenin antibody was either omitted or replaced by an incubation
with non-immune sheep serum.
Double hybridization reaction
Chemiluminescence ISH can also be used for the simultaneous detection
of different DNA sequences in the same sample, utilizing both HRP and
AP as reporter enzymes (Gentilomi et aI., 1997). In the double hybridization reaction, different probes to different targets (e.g. cytomegalovirus
(CMV) DNA and Herpes simplex DNA) have to be labeled with indirect
labels individually detectable by systems with covalently attached AP or
HRP signaling groups.
The double hybridization reaction for the simultaneous detection of
HSV DNA and CMV DNA in the same sample is performed as follows:
Overlay dehydrated cells/sections with 20 III of the hybridization mixture (see above), containing both 2 ng/1l1 ofbiotin labeled HSV DNA probe
and 2 ng/1l1 of digoxigenin-Iabeled CMV DNA probe.
Put cells/sections to hybridize and then wash them under stringent
conditions as described above for single hybridization.
Wash cells/sections briefly in a 150 mM Tris HCI buffer, pH 7.5, containing 150 mM NaCl.
Incubate cells/sections for 30 min at room temperature with a mixture of
streptavidin-HRP complex (NEN-DuPont, Boston, MA,USA) diluted 1/500
in blocking buffer (Boehringer) and polyc1onal anti-digoxigenin Fab fragments conjugated to AP (diluted 1/500 in blocking buffer) (Boehringer).
Wash 3 times for 5 min each in TNT buffer (0.1 MTris HCI, pH 7.5; 0.15
M NaCI; 0.05% Tween 20). Perform the chemiluminescent detections of
the two probes sequentially:
for the HSV DNA probe which utilized HRP as a reporter enzyme, add
to the cells/sections 20-40 JlI of luminol-based chemiluminescent to substrate (e.g. ECL system: luminollHzOz/enhancer) prepared according
to the manufacturer's instructions and measure the light output after
2-4 min incubation;
for the CMV DNA probe which utilized AP as a reporter enzyme, wash
three times for 2 min each in equilibration buffer (l00 mM Tris HCI, 100
355
No luminescent signal should be detectable in target positive cells/sections after hybridization with unlabelled probes, followed by the immunoenzymatic chemiluminescent treatment.
Target positive cells/sections must be completely negative after hybridization with the labeled probes when the primary incubation with antidigoxigenin antibody was either omitted or replaced by an incubation
with non-immune sheep serum.
Double hybridization reaction
Chemiluminescence ISH can also be used for the simultaneous detection
of different DNA sequences in the same sample, utilizing both HRP and
AP as reporter enzymes (Gentilomi et aI., 1997). In the double hybridization reaction, different probes to different targets (e.g. cytomegalovirus
(CMV) DNA and Herpes simplex DNA) have to be labeled with indirect
labels individually detectable by systems with covalently attached AP or
HRP signaling groups.
The double hybridization reaction for the simultaneous detection of
HSV DNA and CMV DNA in the same sample is performed as follows:
Overlay dehydrated cells/sections with 20 III of the hybridization mixture (see above), containing both 2 ng/1l1 ofbiotin labeled HSV DNA probe
and 2 ng/1l1 of digoxigenin-Iabeled CMV DNA probe.
Put cells/sections to hybridize and then wash them under stringent
conditions as described above for single hybridization.
Wash cells/sections briefly in a 150 mM Tris HCI buffer, pH 7.5, containing 150 mM NaCl.
Incubate cells/sections for 30 min at room temperature with a mixture of
streptavidin-HRP complex (NEN-DuPont, Boston, MA,USA) diluted 1/500
in blocking buffer (Boehringer) and polyc1onal anti-digoxigenin Fab fragments conjugated to AP (diluted 1/500 in blocking buffer) (Boehringer).
Wash 3 times for 5 min each in TNT buffer (0.1 MTris HCI, pH 7.5; 0.15
M NaCI; 0.05% Tween 20). Perform the chemiluminescent detections of
the two probes sequentially:
for the HSV DNA probe which utilized HRP as a reporter enzyme, add
to the cells/sections 20-40 JlI of luminol-based chemiluminescent to substrate (e.g. ECL system: luminollHzOz/enhancer) prepared according
to the manufacturer's instructions and measure the light output after
2-4 min incubation;
for the CMV DNA probe which utilized AP as a reporter enzyme, wash
three times for 2 min each in equilibration buffer (l00 mM Tris HCI, 100