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FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ON HUMAN CHROMOSOMES AND INTERPHASE NUCLEI protocol 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 37C in 100g/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 37C (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/l
. 1l labeled probe (10ng/l)
+ 9l 60% formamide/2x SSCP
single copy probes . final concentration = 4ng/l
. 2l labeled probe (20ng/l)
+ 8l 50% formamide/2x SSCP

simultaneous denaturation of probe and target DNA:

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

. overnight hybridisation in moist chamber at 37C (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 4C

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 (10l/slide)

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

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

10. chill immediately on ice for 2-3 min

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

12. denature slides with 100l 70% formamide/2x SSCP 2.5 min (under coverslip) (hot plate 80C) 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 (40C) and apply 10l of preannealed probe under coverslip (18x18mm)

16. overnight hybridization in moist chamber at 37C
(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 42C (cosmid, single copy, library, YAC) to remove excess probe.

wash 5x2 min 0.1xSSC at 42C (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 100l of 0.05% NFDM in moist chamber [ FTCH (for two color hybridization) incubate in 100l 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 100l/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 37C [(b) single copy]

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

. incubate 20 min with 100l/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 37C [( b) single copy]

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

. incubate 20 min with 100l/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 37C [(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 (35l) (DABCO+PI)

for digoxigenated centromere probes, cosmids, YACs and libraries

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

. incubate 20 min with 100l/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 37C

. 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 42C

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

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

incubate 20 min with 100l/slide of 1:200 diluted Neutralite-avadin-FITC (5% NFDM solution or for FTCH 0.5%Blocking reagens solution) at 37C . 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

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

1. x l bidestilized water till endvolume is 50 l

5 l 10x/Nick translationbuffer

5 l nucleotidenmix : 3 l stock solution + 27 l bidest(1)
stock concentration=100mM
nucleotiden : dATP,dGTP,dCTP
25 l of each (1)+25 l H20=100 l mix
endconcentration=2.5 mM

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

5 l 100mM DTT

x l DNA (usual 1g disolved in 1l TE)

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

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

2. mix carefully with finger
2 hr at 15C (cryostat)

for Yacs 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 l 0.5mM EDTA(pH 7.4)

4. to each tube add :
2.5 l salmon sperm DNA(stock 10mg/ml)
2.5 l yeast RNA
5.5 l 3 M NA-acetate (pH5.6)
137.5 l icecold 100% ETOH

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

7. centrifuge 30 min at 14000 rpm at 4C

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

-repetitive probes in 100 l 60% form/SSCP

-unique sequences in 50 l 50% form/SSCP

-cosmids, YACS in 50 l TE

-libraries in 100 l TE