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to maximum, recede and then evaporate. To clean up any cytoplasm we
have found that effective results are achieved if a fresh drop of fixative is
allowed to fall onto the spot at the point when the spreading fixative has
reached its maximum. Allow the drop of fixative to evaporate and re-examine
the spot.
d)
Spotting of the slide. Pipette 4µl of cell suspension onto all 8 areas of the
template slide in a sequence of alternating squares as shown below. This
will prevent the cell spreads from interfering with each other.
e)
Once the first group of drops has air-dried, spot the remaining squares with
4µl drops in the same manner. After the slide has dried, examination of the
slide under phase contrast will reveal whether any squares have been
missed. If spots have been missed, or squares have too few cells, simply
spot those squares again: it is not necessary to re-spot a new slide. If upon
examination of the slide, a square has insufficient cells/metaphases, further
drop(s) of suspension can be added to increase the cell density.
Please note: If the metaphases appear overspread, then clean a new template
slide thoroughly in methanol and re-spot allowing every spot to dry before
proceeding to the next.
2.
Preparation of the Chromoprobe Multiprobe
slide
a)
Ensure that the Chromoprobe Multiprobe
37ºC water bath and allow it to equilibrate to 37ºC (+/- 1ºC). This may take
up to an hour if the water bath has been switched on from cold.
b)
Mix the hybridisation solution by repeated pipetting and pre-warm a 25µl
aliquot per device to 37ºC. Also pre-warm each device by placing it on a
37ºC hotplate label side down. Do not touch the raised surfaces of the
device.
c)
Immerse template slides containing fixed samples in 2xSSC for 2 minutes
at room temperature (RT) without agitation.
d)
Whilst the device is still at 37ºC, dehydrate template slides containing fixed
samples through an ethanol series (2 minutes each in 70%, 85% and 100%)
at RT, air dry and place at 37ºC hotplate to warm up.
e)
Add 2µl of pre-warmed hybridisation solution to each of the eight areas on
the pre-warmed device using a P10 micropipette while it remains on a 37ºC
hotplate.
3.
Positioning of template slide over the device
a)
Carefully invert the template slide over the device such that the number 1,
which is now upside down, is located over the top right hand area of the
device. To help locate square 1 (Chromosomes 3, 15 & 17), its position on
the device has been marked with an orange dot).
b)
Make sure that the template slide is carefully aligned with the matching
areas on the device. Carefully lower the slide over the device so that the
drops of hybridisation solution make contact with the slide. Apply gentle,
even pressure to ensure that the hybridisation solution is spread to the
edges of each of the raised areas on the device.
c)
Lift the slide carefully holding the frosted end of the glass slide and invert so
that the template slide is underneath the device. Make sure the device does
not smear across the template slide as this could cause cross-contamination
of the probes.
d)
Place at 37ºC (+/- 1ºC) (hotplate or incubator) for 10 minutes.
4.
Instructions for use of the Cytocell Slide Surface Thermometer
a)
The temperature of the 75ºC hotplate should be checked with the Cytocell
Slide Surface Thermometer before proceeding to denaturation.
b)
To use the thermometer properly, place it onto the surface of the hotplate
and wait until the different segments stop changing colour. The actual
temperature is indicated by a deep aqua colour.
Please note:
c)
When the segments appear granular and the colours no longer appear
uniform and regular, the thermometer should be discarded as it is
exhausted. The life span of each thermometer should, however, easily be
sufficient for a ten-device kit.
d)
This thermometer is a liquid crystal device and although reusable, it must be
treated with care to ensure a reasonable life span. The thermometer must
only be used to check the temperature of a hotplate; it must not be used to
monitor the hotplate performance over time.
5.
Denaturation
Please note: A PCR thermal cycler-heating block is NOT suitable for use in
place of solid bed hotplate for this procedure.
a)
Transfer the slide/device sandwich to the hotplate taking particular care to
hold it level. Ensure the sample slide is in good contact with the hotplate.
b)
Denature on the hotplate at 75ºC (+/- 1ºC) for 5 minutes.
®
device and template
®
Hybridisation Chamber is in the
6.
Hybridisation
Place the slide/device sandwich in the pre-warmed Chromoprobe
®
Multiprobe
Hybridisation Chamber, replace the lid and float the chamber in
the 37ºC (+/- 1ºC) water bath (non-stirring) overnight.
Please note:
a)
Do not seal the lid on the hybridisation chamber.
b)
Do not place a lid on the water bath.
c)
Do not hybridise in an incubator.
d)
Please ensure that the hybridisation chamber is completely dry (i.e. no
water or damp tissue inside the chamber).
The humidity inside the chamber is vital for optimal hybridisation. The
correct levels will be achieved following those steps.
7.
Post-hybridisation stringent washes
Please note: Avoid processing more than two slides through the stringency
washes at any one time.
a)
Remove the device carefully from the slide.
b)
Immerse the slide in 0.4xSSC (pH 7.0) at 72ºC (+/- 1ºC) for 2 minutes
without agitation.
c)
Drain the slide and immerse it in 2xSSC, 0.05% Tween-20 at RT (pH 7.0)
for 30 seconds without agitation.
8.
Mounting and visualisation of results
a)
Drain the slide and apply 20µl of DAPI antifade to each end of the slide.
b)
Cover with a coverslip (24x50mm or 24x60mm), remove any bubbles and
allow the colour to develop in the dark for 10 minutes.
c)
View with a fluorescence microscope.
Please note: Certain types of microscope have slide holders, which make it
difficult to view the extreme ends of the slide. If this occurs then simply turn the
slide through 180, which will help with the viewing of the slide.
Stability of Finished Slides
FISHed slides remain analysable for up to 1 month if stored in the dark at/or
below RT.
Procedural Recommendations
1.
Baking or ageing of slides may reduce signal fluorescence
2.
Hybridisation conditions may be adversely affected by the use of reagents
other than those provided or recommended by Cytocell Ltd.
3.
Use a calibrated thermometer for measuring temperatures of solutions,
waterbaths and incubators as these temperatures are critical for optimum
product performance.
4.
The wash concentrations, pH and temperatures are important as low
stringency can result in non-specific binding of the probe and too high
stringency can result in a lack of signal.
5.
Incomplete denaturation can result in lack of signal and over denaturation
can also result in non-specific binding.
6.
Over hybridisation can result in additional or unexpected signals.
7.
Users should optimise the protocol for their own samples prior to using the
test for diagnostic purposes.
8.
Slides should be independently scored by two analysts.
9.
Suboptimal conditions may result in non-specific binding that may be
misinterpreted as a probe signal.
Expected Results
1.
The acrocentric chromosome painting probes contain short arm material.
This is shared between the D & G group chromosomes, so cross-
hybridisation in these regions may be observed.
2.
The chromosome 16 whole chromosome painting probe may show faint
cross-hybridisation to the heterochromatic regions of chromosome Y.
3.
The Y chromosome painting probe contains the pseudoautosomal regions
common with the X chromosome. Consequently, cross-hybridisation may be
observed in these regions.
4.
Chromosomes 1, 5 and 19 have centromeric DNA sequences in common.
Consequently cross-hybridisations in these regions may be observed
between these chromosomes.
5.
Chromosome 1 whole chromosome painting probe may show a brighter
signal at 1p36.
6.
Whole chromosome painting probes do not cover the Heterochromatic
blocks in chromosomes 1, 9 and 16.
Limitations
Reporting and interpretation of FISH results should be consistent with
professional standards of practice and should take into consideration other
clinical and diagnostic information. This kit is intended as an adjunct to other
diagnostic laboratory tests and therapeutic action should not be initiated on the
basis of the FISH result alone.
Failure to adhere to the protocol may affect the performance and lead to false
positive/negative results.
This kit has not been validated for purposes outside of the intended use stated.
Additional Information
For additional product information please contact the Cytocell Technical
Support Department.
T: +44 (0)1223 294048
E: techsupport@cytocell.com
W: www.cytocell.com
L'hybridation in situ par fluorescence (FISH) est une technique qui permet de détecter des
séquences ADN sur les chromosomes en métaphase ou sur les noyaux interphasiques
FRANÇAIS
PI008/CE v011.00/2019-04-30
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