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FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ON HUMAN CHROMOSOMES AND INTERPHASE NUCLEI

Dept of Medical Genetics
University Hospital Gent
B-9000 Gent Belgium

SLIDE PRETREATMENT

a. RNase pretreatment

. incubate 1 hour at 37°C in 100µg/ml RNaseA/2xSSC pH 7 in moist chamber (incubator)
. wash 3x5 min with 2xSSC
. dehydration in ethanol series (70%, 90%, 94%)
. air dry (and store dust free)

b. pepsin treatment

. incubate 30 min at 37°C (WWB) in 0.005% pepsin/0.01 M HCl (stock concentration pepsin=10% in H2O)
. rinse with 1xPBS

c. postfixation

. prewash 5 min at RT in postfixation buffer
(100ml : 10ml 10xPBS + 10ml 0.5 M MgCl2 + 80ml bidest)
. 5 min postfixation in 4% paraformaldehyde at RT
(100ml : 10ml 10xPBS + 10ml 0.5 M MgCl2 + 30ml bidest + 50ml 8%PFA)
. wash in 1xPBS 5min at RT
. dehydratation in ethanol series (70%, 90%, 94%) and air dry

PROBE PREPARATION

A. PLASMID PROBES

centromeric probes . final concentration = 1ng/µl

. 1µl labeled probe (10ng/µl)
+ 9µl 60% formamide/2x SSCP

single copy probes . final concentration = 4ng/µl

. 2µl labeled probe (20ng/µl)
+ 8µl 50% formamide/2x SSCP

simultaneous denaturation of probe and target DNA:

apply 10µl of the probe mixture under a coverslip (24*24mm) and denature 5 min at 80°C (hot plate)

. overnight hybridisation in moist chamber at 37°C (incubator)

B. COSMID-, YAC- and LIBRARY PROBES

1. add 50x excess COT-I-DNA to 20ng/ slide (cosmids, YAC's) or 100ng/ slide (libraries)

2. precipitate with Na-Acetate (3 M pH 5.6; 1/10 of volume of DNA) and ice-cold ethanol 100% (2.5x volume DNA)

3. chill on ice for 30 min

4. centrifuge 30 min at 14000 RPM 4°C

5. remove supernatans

6. air-dry pellet for 15 min

7. dissolve the pellet in an appropriate volume of 50% formamide/12.5% dextransulphate/2x SSCP (10µl/slide)

8. incubate 30 min at 37°C (WWB) to dissolve the pellet completely

9. denature probes at 70°C (WWB) for 5 min

10. chill immediately on ice for 2-3 min

11. allow probes to preannealing at 37°C (WWB) for 5-30 min

12. denature slides with 100µl 70% formamide/2x SSCP 2.5 min (under coverslip) (hot plate 80°C) during the last 15 min of the preannealing

13. remove coverslip and chill slides in ice-cold 70% ethanol and rinse for 2.5 min followed by rinses in 90% and 94% ethanol

14. dry slides on a hot plate (40°C) and apply 10µl of preannealed probe under coverslip (18x18mm)

16. overnight hybridization in moist chamber at 37°C
(incubator)

DAY 2

preparation of wash-solutions

-50% formamide/2xSSC pH 7.0
-0.1xSSC
-2xSSC
-4xSSC/Tween 0.05%
-5% NFDM (non fat dry milk) in 4xSSC
-for digoxigenin labeled probes prepare 0.15 M NaCl+0.1 M tris-HCl/Tween 0.05% and 0.5% Blocking reagens (Boehringer) in 0.1M tris+0.15 M NaCl

PROBE DETECTION

. wash 3x5 min with 50% formamide/2xSSC at RT (centr. probes) or at 42°C (cosmid, single copy, library, YAC) to remove excess probe.

wash 5x2 min 0.1xSSC at 42°C (cosmid,single copy, library, YAC) or 2x5 min at RT (centr. probes) with 2xSSC

. wash 1x5 min 2xSSC for all slides at RT

. wash 1x5 min 4xSSC/Tween RT or for bio+dig or dig only wash 1x5 min 0.15M NaCl+0.1M Tris/Tween

. blocking step: incubate 10 min (centr.probes, single copys) or 20 min (cosmids, libraries, YACS) at RT with 100µl of 0.05% NFDM in moist chamber [ FTCH (for two color hybridization) incubate in 100µl of 0.5% Blocking reagens]

. wash 1x5 min with 4xSSC/Tween or for FTCH 0.15M NaCl+0.1 M Tris/Tween at RT

immunocytochemical detection :

. incubate 20 min with 100µl/slide of 1:200 diluted Neutralite-avidin-FITC (5% NFDM solution or for FTCH 0.5% Blocking reagens solution) in moist in dark at RT [(a) centr., cosmids, YACS, libraries] or at 37°C [(b) single copy]

. wash 3x5 min in 4xSSC/Tween or for FTCH tris-NaCl-buffer at RT (a) or at 42°C (b)

. incubate 20 min with 100µl/slide of 1:100 diluted biotinylated goat-anti-avidin (5% NFDM solution) + for FTCH 1:500 diluted mouse anti-digoxin (0.5% Blocking reagens solution) in moist in dark at RT [(a) centr., cosmids, YACS, libraries] or at 37°C [( b) single copy]

. wash 3x5 min in 4xSSC/Tween or for FTCH tris-NaCl-buffer at RT (a) or at 42°C (b)

. incubate 20 min with 100µl/slide of 1:200 diluted Neutralite-avidin-FITC (5% NFDM solution) + for FTCH 1:100 sheep anti-mouse digoxigenin (0.5% Blocking reagens solution) in moist in dark at RT [(a) centr., cosmids, YACS, libraries] or at 37°C [(b) si ngle copy]

for biotinylated centromere probes, cosmids, YACs and libraries

. wash2x5 min in 4xSSC/Tween

. 1x5 min in 1xPBS

. deshydratation in ethanol series

. air dry and mount in mount medium (35µl) (DABCO+PI)

for digoxigenated centromere probes, cosmids, YACs and libraries

. wash 3x5 min in tris-NaCl-buffer at RT

. incubate 20 min with 100µl/slide of 1:100 diluted sheep anti-digoxigenin-TRITC (0.5% Blocking reagens solution) in moist in dark at RT

. wash 2x5 min in tris-NaCl-buffer at RT

. 1x5 min in 1xPBS

. deshydratation in ethanol series

. air dry and mount in mount medium (DAPI)

for FTCH

1:100 diluted sheep anti-digoxigenin-TRITC (0.5% Blocking reagens solution) in moist in dark at 37°C

. 1x5 min in 1xPBS

. deshydratation in ethanol series

. air dry and mount in mount medium (DAPI)

for single copies

. wash 3x5 min in 4xSSC/Tween or for FTCH tris-NaCl-buffer at 42°C

. incubate 20 min with 100µl/slide of 1:100 diluted biotinylated goat-anti-avidin (5% NFDM solution) in moist dark at 37°C

. wash 3x5 min in 4xSSC/Tween or for FTCH tris-NaCl-buffer at 42°C

incubate 20 min with 100µl/slide of 1:200 diluted Neutralite-avadin-FITC (5% NFDM solution or for FTCH 0.5%Blocking reagens solution) at 37°C . wash 2x5 min in 4xSSC/Tween or for FTCH in tris-NaCl-buffer at RT

. 1x5 min in 1xPBS

. deshydratation in ethanol series

. air dry and mount in mount medium (DABCO+PI) (DAPI for FTCH)

Nick translation of dsDNA with biotinylated or digoxigenin dUTP

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For the use with FISH (fluorescent in situ hybridization) we always label the entire plasmid and do not cut out the insert.

1. x µl bidestilized water till endvolume is 50 µl

5 µl 10x/Nick translationbuffer

5 µl nucleotidenmix : 3 µl stock solution + 27 µl bidest(1)

stock concentration=100mM
nucleotiden : dATP,dGTP,dCTP
25 µl of each (1)+25 µl H20=100 µl mix
endconcentration=2.5 mM

2.5 µl bio-16-dUTP(0.4mM,ready for use)

5 µl 100mM DTT

x µl DNA (usual 1µg disolved in 1µl TE)

5 µl DNase I (stock 1mg/ml,dilute 1/1000 in H20, prepare fresh every time)

x µl DNA polymerase (endconcentration must be=30 U)

2. mix carefully with finger

2 hr at 15°C (cryostat)

for Yac´s check fragment length on 1,2% agarose gel. The fragment length should be 300-400kB. If the fragment length is smaller add more DNase I.

3. stop the reaction with 5 µl 0.5mM EDTA(pH 7.4)

4. to each tube add :

2.5 µl salmon sperm DNA(stock 10mg/ml)
2.5 µl yeast RNA
5.5 µl 3 M NA-acetate (pH5.6)
137.5 µl icecold 100% ETOH

6. this mixture overnight or 1 hr at -70°C (precipitation)

7. centrifuge 30 min at 14000 rpm at 4°C

8. remove supernatans (vacuumpomp) and dissolve in appropiate volume :

-repetitive probes in 100 µl 60% form/SSCP

-unique sequences in 50 µl 50% form/SSCP

-cosmids, YACS in 50 µl TE

-libraries in 100 µl TE

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