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MONICA MUSIANI et al.
enhancer) prepared following the manufacturer's instructions over the
sample on the glass slide, and put the slide under the microscope connected with the video camera.
Acquire the light signal in a few (between two and five) minutes. In this
case, the light emission kinetics is ofa glowing type with a plateau stable for at
least 10 min and whose intensity is proportional to the enzyme activity.
To detect AP-Iabeled hybridized probes: Wash cells/sections for 2 min
with equilibration buffer (EB) (100 mM Tris HCI, 100 mM NaCl, 50 mM
MgCh, pH 9.5). Add a 20-40 JlI drop of dioxetane phosphate-based reagents (Le. CSPD, CDP-star, Lumi-Phos Plus) over the sample.
Acquire the light emission after 20 min of incubation. The kinetics in
this case, is also of a glowing type, but it is slower and about 15 min is the
necessary time to reach a steady-state light emission.
Quantitative analysis
A control of uninfected cells and a negative biopsy specimen provide the
threshold background levels. Threshold values are calculated analysing a
mean of 50 cells from control negative samples when cellular smears have
to be tested, and 10 microscopic fields from negative reference biopsies
when a biopsy specimen has to be analyzed. The average SD values of the
background light emission (expressed as photons/second/area) is then
calculated. The average value of the background signal plus five-fold
its standard deviation is considered the threshold value above which
the chemiluminescent signal resulting from the hybridized nucleic acid
can be determined as positive. The net light signal of the sample is obtained automatically on the screen after subtraction of the threshold values using the appropriate software.
Specificity controls for chemiluminescence in situ hybridization
The following control experiments are necessary to prove that the chemiluminescence hybridization reaction is detecting target sequences specifically.
A specific positive signal is to be detected when reference samples of
target positive cells are treated with labeled probes.
No chemiluminescent signal should be observed when target positive
cells/sections are hybridized with vector control DNA labeled probe and
treated with anti digoxigenin Fab fragment conjugated with the enzyme
and with the chemiluminescent substrate.
MONICA MUSIANI et al.
enhancer) prepared following the manufacturer's instructions over the
sample on the glass slide, and put the slide under the microscope connected with the video camera.
Acquire the light signal in a few (between two and five) minutes. In this
case, the light emission kinetics is ofa glowing type with a plateau stable for at
least 10 min and whose intensity is proportional to the enzyme activity.
To detect AP-Iabeled hybridized probes: Wash cells/sections for 2 min
with equilibration buffer (EB) (100 mM Tris HCI, 100 mM NaCl, 50 mM
MgCh, pH 9.5). Add a 20-40 JlI drop of dioxetane phosphate-based reagents (Le. CSPD, CDP-star, Lumi-Phos Plus) over the sample.
Acquire the light emission after 20 min of incubation. The kinetics in
this case, is also of a glowing type, but it is slower and about 15 min is the
necessary time to reach a steady-state light emission.
Quantitative analysis
A control of uninfected cells and a negative biopsy specimen provide the
threshold background levels. Threshold values are calculated analysing a
mean of 50 cells from control negative samples when cellular smears have
to be tested, and 10 microscopic fields from negative reference biopsies
when a biopsy specimen has to be analyzed. The average SD values of the
background light emission (expressed as photons/second/area) is then
calculated. The average value of the background signal plus five-fold
its standard deviation is considered the threshold value above which
the chemiluminescent signal resulting from the hybridized nucleic acid
can be determined as positive. The net light signal of the sample is obtained automatically on the screen after subtraction of the threshold values using the appropriate software.
Specificity controls for chemiluminescence in situ hybridization
The following control experiments are necessary to prove that the chemiluminescence hybridization reaction is detecting target sequences specifically.
A specific positive signal is to be detected when reference samples of
target positive cells are treated with labeled probes.
No chemiluminescent signal should be observed when target positive
cells/sections are hybridized with vector control DNA labeled probe and
treated with anti digoxigenin Fab fragment conjugated with the enzyme
and with the chemiluminescent substrate.