Islamic University of Gaza
Fluorescent In-Situ Hybridization
FISH
Training Course Manual
Dr. Basim Ayesh
August, 2009
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Procedure for FISH analysis of chromosomes 13/21/X/Y/18
Reagents:
Poseidon™ Repeat Free™ Chromosome 13/21, X/Y/18 specific DNA Probes
Vial 1
 Critical region 1 (red): The 21q specific DNA probe is direct-labeled with
PlatinumBright550.
 Critical region 2 (green): The 13q14 specific DNA probe is direct-labeled with
PlatinumBright495.
Vial 2
 Critical region 3 (blue): The 18 SE DNA probe is direct-labeled with
PlatinumBright415.
 Critical region 4 (green): The X SE DNA probe is direct-labeled with
PlatinumBright495.
 Critical region 5 (red): The Y SE DNA probe is direct-labeled with PlatinumBright550.
Intended use:
The chromosome 21 specific region probe is optimized to detect copy numbers of chromosome
21 at 21q22.1 on uncultured amniotic cells.
The chromosome 13 specific region probes is optimized to detect copy numbers of
Chromosome 13 at 13q14.2 on uncultured amniotic cells.
The chromosome 18 specific Satellite probe (D18Z1) is optimized to detect copy numbers of
Chromosome 18 at 18p11-18q11 on uncultured amniotic cells.
The chromosome X specific Satellite probe (DXZ1) is optimized to detect copy numbers of
Chromosome X at Xp11-Xq11 on uncultured amniotic cells.
The chromosome Y specific Satellite probe (DYZ3) is optimized to detect copy numbers of
Chromosome Y at Yp11-Yq11 on uncultured amniotic cells.
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Training session Day 2
The class will be divided into groups of two trainees each. Each group will prepare 2 slides
from uncultured blood.
 Three interphase slide-preparations (labeled 1, 1a and 1b) will be selected from the
prepared uncultured blood slides (for 13/21 probe mix).
 Three interphase slide-preparations (labeled 2, 2a and 2b) will be selected from the
prepared uncultured blood slides (for X/Y/18 probe mix).
Specimen:

Uncultured blood and bone marrow preparations for interphase FISH:
Sample preparation:
1. Add 10 ml of 0.075 mM KCL to 0.25-1 ml of blood or bone marrow in a centrifuge
tube.
2. Incubate for 15 min at 37°C.
3. Centrifuge for 5 min.
4. Remove supernatant and slowly add 5 ml fixative while vortexing.
5. Centrifuge and replace the fixative two to three times or until a clear cell
suspension is observed.
Slide making:
1. Clean microscope slides by dipping in methanol and drying by wiping with lintfree cloth to ensure the slides are grease-free.
2. Centrifuge the fixed cell suspension for 5 min at 200 xg in a swinging bucket
rotor.
3. Remove the supernatant.
4. Add a drop of the suspension onto a microscope slide.
5. Allow to air dry.
6. Check the cell density under phase contrast microscope.

Heparinized whole blood cultured in RPMI 1640 medium supplemented with fetal
bovine serum, penicillin, streptomycin and L-glutamine and 2% PHA. The blood cultures
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are harvested and fixed by methanol/acetic acid and the slides prepared according to
standard techniques (refer to materials from karyotyping training session).
 The trainers will demonstrate the procedure for the trainees on slide # 1 and 2.
 The trainees will be divided into 4 groups (1a, 1b, 2a,2b).
Slides pretreatment:
1. Fill a vertical Coplin jar with 50 ml pretreatment buffer (see preparations).
2. Incubate the jar at 37°C for enough time to worm the pretreatment buffer, before
proceeding with the procedure
3. Dip the prepared slides into the pre-wormed buffer
4. Incubate at 37°C for 15 min.
Dehydration:
1. During the incubation period prepare 3 horizontal Coplin jars containing 100 ml of the
following ethanol concentrations at room temperature: (70%, 85% and 100%).
a. Dip the slides in 70% ethanol for 1 min
b. Dip the slides in 85% ethanol for 1 min
c. Dip the slides in 100% ethanol for 1 min
d. Air-dry the slides.
Co-Denaturation/Hybridization:
1. Apply 10μl of 13/21 probe (ready to use) onto each of slides #1 and 1a. Avoid
generation of air bubbles.
2. Apply 10μl of X/Y+18 probe preparation or (ready to use) onto each of slides # 2 and
2a. Avoid generation of air bubbles.
3. Gently cover each slide with (22 X 22 mm) cover slip, and make sure that the applied
probe preparation is uniformly spread beneath the cover slips.
4. Seal the cover slips to the slides with Fixogum.
5. Incubate the slides at 75°C for 5-10 min on a hotplate with precise temperature control
(Thermal cycler may be used).
6. Incubate the slides in a sealed humidified box at 37°C for overnight (12-16 Hrs).
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Training session Day 3
Post-Hybridization stringency washing:
1. Prepare two Coplin jars:
a. Fill the first with 100 ml of Post-wash buffer I, and pre incubate in a water bath
adjusted at 72°C for enough time to raise the buffer temperature to the desired
temperature (72°C).
b. Fill the second jar with 100 ml of Post-wash buffer II and keep at room
temprater.
2. Remove the Fixogum seal.
3. If necessary incubate the slides in Post-wash buffer II for 2 min at room temperature to
slide off the cover slips.
4. Incubate the slides in a Coplin jar containing pre-wormed (72°C) Post-wash Buffer I for 2
min.
5. Wash slides in Post- wash Buffer II for 1 min at room temperature.
6. Dehydrate the slides for 1 min in each of: 70 %, 85 % and 100 % ethanol.
7. Air-dry at room temperature.
Counter-staining:


Apply 15 μl of DAPI/antifade and apply a glass cover slip
Visualize by a fluorescent microscope.
Interpretation:
The Chromosome 13/21 specific probe is designed as a dual-color assay to detect gains of
chromosome 21 and 13. Trisomy 21 will be detected by three red signal at the 21q22 region
and two green signals for chromosome 13 (3R2G). Trisomy 13 will be detected by 3 green
signals at the 13q14 region and two red signals for chromosome 21 (2R3G).
Two single color red (R) and green (G) signals will identify the normal chromosomes 13 and 21
(2R2G).
The Chromosome X/Y/18 specific probe is designed as a triplecolour assay to detect gains or
losses of chromosome X, Y and or 18. Turner syndrome will be detected by one green signal
only at Xcen. Meta-Females (or Triple-X females) will be detected by three or more green
signals at Xcen. Klinefelter will be detected by 2 or more green and 1 red signal. XYY males will
be detected by one green and two red signals. Two single green (G) signals will identify the
normal X chromosome in females, one green and one red signal will identify the normal X and Y
chromosomes in male. Trisomy 18 will be detected by three blue signals at 18 cen.
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Two single blue signals will identify the normal chromosome 18.
Interpretation Table:
Normal Signal
Pattern
Trisomy 21
Trisomy 13
2R2G
3R2G
2R3G
3R2G2B
2R3G2B
Expected Signals
Using 13/21
Expected Signals
Using 13/21+18
Expected Signals
Using X/Y + 18
Expected Signals
Using X/Y
Expected Signals
Using X/Y + 18
2R2G2B
Trisomy 18
2R2G3B
Female
Male
Female
Male
2G2B
1R1G2B
2G3B
1R1G3B
Female
Male
2G
2G2B
1R1G
1R1G2B
Turner XO
1G
1G2B
Meta-female
Klinefelter
XYY
3-5G
2G1R
3-4G1R
1R1G/1R2G in
mosaics
1G2R
3-5G2B
2G1R2B
3-4G1R2B
1R1G2B/1R2G2B in
mosaics
1G2R2B
Trisomy 21:
One of the most common chromosomal abnormalities in live born children and causes
Down syndrome, a particular combination of phenotypic features that includes mental
retardation and characteristic facies. Molecular analysis has revealed that the 21q22.1q22.3 region appears to contain the gene(s) responsible for the congenital heart disease
observed in Down syndrome.
Trisomy 13:
Also called Patau syndrome, is a chromosomal condition that is associated with severe
mental retardation and certain physical abnormalities. The critical region has been reported
to include 13q14-13q32 with variable expression, gene interactions, or interchromosomal
effects.
Trisomy 18:
Causing Edwards syndrome is the second most common autosomal trisomy after
trisomy 21. The disorder/condition is characterized by severe psychomotor and growth
retardation, microcephaly, microphthalmia, malformed ears, micrognathia or retrognathia,
microstomia, distinctively clenched fingers, and other congenital malformations.
Chromosomal abnormalities involving the X and Y chromosome (sex chromosomes) are
slightly less common than autosomal abnormalities and are usually much less severe in
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their effects. The high frequency of people with sex chromosome aberrations is partly due
to the fact that they are rarely lethal conditions.
Turner syndrome:
Occurs when females inherit only one X chromosome; their genotype is X0.
Metafemales or triple-X females:
Inherit three X chromosomes; their genotype is XXX or more rarely XXXX or XXXXX.
Klinefelter syndrome:
Males inherit one or more extra X chromosomes; their genotype is XXY or more rarely XXXY,
XXXXY, or XY/XXY mosaic.
XYY syndrome:
Males inherit an extra Y chromosome; their genotype is XYY.
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Buffers and preparations:
Training session Day 2
1. Fixative:
Methanol
Glacial acetic acid
Total
2.
30 ml
10 ml
40 ml
Ptassium chloride:
0.075 mM
3. Pretreatment buffer:
20 X SSC buffer
Triton-X-100
Distilled Water
Total
4.
5 ml
250 μl
45 ml
50 ml
Probe preparation: (in case not ready to use)
Hybridization Buffer
Probe
Total
13/21 probe mix
X/Y + 18 probe mix
ready to use
8 μl
Training session Day 3
5.
10 X wash buffer I
Distilled water
Post-Wash buffer I:
5 ml
45 ml
Total
50 ml
10 X wash buffer I
20 X SSC
Distilled water
Post-Wash buffer II:
2 ml
5.6 ml
52.5 ml
Total
50 ml
6.
8
2 μl
10 μl
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