28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
353
Hybridization reaction
Incubate cells/sections with 10 III of the hybridization mixture (see above).
A clean glass coverslip is applied and the edges are sealed with nail polish
to prevent loss of the mixture during denaturation and hybridization.
Cells/sections and the hybridization mixture containing the labeled probe
are denatured together by heating in an 85°C water bath for 6 min, and are
then hybridized at 37°C for 3 h.
The coverslips are carefully removed and cells/sections are washed at
stringent conditions in order to minimize non-specific hybridization
(three times at 34°C, with 50% formamide and 2xSSC for 3 min each,
followed by two 3 min washes in 2x SSC at 34°C and finally one 3 min
wash in 2xSSC at room temperature).
Chemiluminescent detection
Wash cells/sections briefly in a 150 mM Tris HCl buffer, pH 7.5, containing
150 mM NaCl. Incubate with the appropriate HRP- or AP-labeled detector
systems. In detail:
a) the samples hybridized with fluorescein labeled probes are incubated
for 30 min at 37°C with HRP- or AP-conjugated Fab fragments to fluorescein isothiocyanate (e.g. HPV probes Dako A/S, Glostrup, Denmark)
used at the dilution suggested by manufacturer's instructions or determined by preliminary block titration;
b) the samples hybridized with digoxigenin labeled probe are incubated
for 30 min at 37°C with HRP- or AP-conjugated anti-digoxigenin Fab
fragments (e.g. HPV probes Kreatech, Amsterdam, The Netherlands)
used at the dilution suggested by the manufacturer's instructions or
determined by preliminary block titration;
c) the samples hybridized with biotinylated DNA probe are incubated for
30 min at 37°C with HRP- or AP-conjugated to streptavidin (e.g. HPV
probes Biohit, Helsinki, Finland and HSV probes Enzo Biochem, New
York, USA) used at the dilution suggested by the manufacturer's instructions or determined by preliminary block titration.
Wash cells/sections three times for 5 min with alSO mM Tris HCl buffer,
pH 7.5 containing 150 mM NaCl.
To detect HRP labeled hybridized probes: Add a 20-40 III drop of luminol-based chemiluminescent substrate (e.g. ECL system: luminollHzO z /
353
Hybridization reaction
Incubate cells/sections with 10 III of the hybridization mixture (see above).
A clean glass coverslip is applied and the edges are sealed with nail polish
to prevent loss of the mixture during denaturation and hybridization.
Cells/sections and the hybridization mixture containing the labeled probe
are denatured together by heating in an 85°C water bath for 6 min, and are
then hybridized at 37°C for 3 h.
The coverslips are carefully removed and cells/sections are washed at
stringent conditions in order to minimize non-specific hybridization
(three times at 34°C, with 50% formamide and 2xSSC for 3 min each,
followed by two 3 min washes in 2x SSC at 34°C and finally one 3 min
wash in 2xSSC at room temperature).
Chemiluminescent detection
Wash cells/sections briefly in a 150 mM Tris HCl buffer, pH 7.5, containing
150 mM NaCl. Incubate with the appropriate HRP- or AP-labeled detector
systems. In detail:
a) the samples hybridized with fluorescein labeled probes are incubated
for 30 min at 37°C with HRP- or AP-conjugated Fab fragments to fluorescein isothiocyanate (e.g. HPV probes Dako A/S, Glostrup, Denmark)
used at the dilution suggested by manufacturer's instructions or determined by preliminary block titration;
b) the samples hybridized with digoxigenin labeled probe are incubated
for 30 min at 37°C with HRP- or AP-conjugated anti-digoxigenin Fab
fragments (e.g. HPV probes Kreatech, Amsterdam, The Netherlands)
used at the dilution suggested by the manufacturer's instructions or
determined by preliminary block titration;
c) the samples hybridized with biotinylated DNA probe are incubated for
30 min at 37°C with HRP- or AP-conjugated to streptavidin (e.g. HPV
probes Biohit, Helsinki, Finland and HSV probes Enzo Biochem, New
York, USA) used at the dilution suggested by the manufacturer's instructions or determined by preliminary block titration.
Wash cells/sections three times for 5 min with alSO mM Tris HCl buffer,
pH 7.5 containing 150 mM NaCl.
To detect HRP labeled hybridized probes: Add a 20-40 III drop of luminol-based chemiluminescent substrate (e.g. ECL system: luminollHzO z /