In-Situ Hybridisation

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In-Situ Hybridisation

In-Situ Hybridisation 1

Detection of Digoxigenin and Biotin Labelled DNA Probes

Using an Immunogold Silver Staining Technique

Numerous non-isotopic systems have now been developed to visualise

labelled gene probes. Antibody based detection methods have proved to be as

sensitive as isotopic labels and provide a suitable alternative for routine in-situ

hybridisation procedures. The use of digoxigenin and biotin labelled DNAprobes coupled with an antibody-enzyme detection method has been shown

to be a useful means of detecting genes and gene expression by in-situ

hybridisation. Here we describe the applications of an alternative detection

system using immunogold conjugates.

In-Situ Hybridisation For Light Microscope

1. Paraffin wax embedded sections were heat fixed onto 3-

aminopropyltriethoxysilane coated slides.2. Sections were de-waxed in xylene (2 x 15 min),re-hydrated through 100%,

70% ethanol and finally washed in deionised water.

3. Pre-treatments included hydrolysis in 0.05M HCl for 10 min followed by

digestion with 20 g/ml proteinase K (in 20mM Tris, 2mM CaCl pH 7.5) for 30

min at 37C. A proteinase titration experiment is necessary for each particular

tissue block to determine optimum digestion conditions.

4. After digestion, sections were washed in l times TBS (0.1M Tris-Cl, 0.15M

NaCl, pH 8.2), post-fixed in a solution of 4% paraformaldehyde for five

minutes, and returned to l times TBS.

5. Sections were placed in pre-hybridisation buffer (50% formamide, 5x

Denhardt's solution, 2x SSC, calf thymus DNA) at 37C for 30 minutes.

6.Double stranded DNA probes were labelled by the multi-prime technique

using commercially available biot in (Vector Labs Inc) and digoxigenin (Roche

Diagnostics) labelling kits. Labelled probe was diluted in hybridisation buffer

(table 1) to between 0.1-0.5 g/ml and applied to section in 10l volume under

a sealed cover slip or under a square of paraffin film.

7. Sections were placed in a moist chamber (Terasaki plate) and probe and


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target DNA were heat denatured simultaneously in an oven at 90C for 10 min.

Hybridisation was carried out overnight at 42C in a moist chamber.

8. Cover slips were removed by soaking slides in 2x SSC containing 0.1%

Tween20.

9. Post-hybridisation washes included, 2x SSC at 20C for 10 min, 2x SSC at

42C for 10 min and twice in 0.5x SSC at 42C for 10 min. Sections were then

equilibrated in lx TBS (0.1M Tris, 0.15M NaCl, pH 8.20).

Detection Of Hybrids With Gold Labelled Antibody

1. After hybridisation sections were blocked in either 5% BSA, 5% normal

sheep serum, or 1% casein, in l times TBS containing 0.1% Tween20, for 1

hour at room temperature.

2. Blocking solution was shaken off and Sheep antiDigoxigenin or Goat anti-

Biotin polyclonal gold conjugates, diluted 1/100 in either 1% BSA or 0.1%

casein in l times TBS containing 0.1% Tween20 were applied to the section

and incubated for 2 hours at room temperature.

3. Sections were washed thoroughly in lx TBS to remove excess antibody.

4. Sections labelled with gold conjugates were washed 5 x 1min in deionised

water.

5. Gold particles were visualised using silver enhancer for approximately 20

min, or until sections appeared optimally developed under light microscope

control. The reaction was stopped by rinsing sections with water.

Pre-Hybridisation Buffer

Item Concentration Volume l/ml Working

Formamide1 500 50%

100x Denhardts2 50 5x

20x SSC3 100 2x

10 mg/ml calf thymus DNA 20 200 g/ml

De-ionised water 30

Probe DNA Upto 300 200 ng/ml

1. Formamide is a teratogen. Handle with extreme care and wear gloves. Todeionise formamide, add 200 ml formamide to 10g of Amberlite MB-3 resin(Sigma). Stir for 1 hour at room temperature. Filter through Whatman No.1 filterpaper to remove the resin. Store at 4C in a dark bott le.


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2. 100x Denhardt's solution is 2% Ficoll (mw 400,000), 2% polyvinylpyrrolidone (mw 400,000) and 2% heat treated BSA. Store at 20C.

3. 1x SSC is 0.l5M NaCl, 0.015M tri-sodium citrate, pH 7.0. Sterilised byautoclaving.


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In-situ Hybridisation 2

Detection of mRNA with Oligonucleotide Probes using an Immunogold Silver

Stain Technique

Short single-stranded oligonucleotide probes are now easily synthesized,

penetrate tissue sections better than larger double-stranded DNA or RNA

probes and can be readily detected with the use of non-isotopic labels. Here

we describe the application of an immunogold antibody detection system to

visualise digoxigenin end labelled oligonucleotide probes.

The gold/silver reaction obtained is viewed as an intense black signal using

bright field microscopy or as a brilliant reflected image under epipolarised

illumination. The reaction product does not fade, is compatible with most

counter stains and mounting agents and individual gold/silver enhanced

particles can be counted under high power magnification to give a quantitative

estimation of gene expression.

Probe Labelling

Probes were labelled at the 3'end using a terminal transferase. The reaction

was carried out as follows:

1. Mix on ice; 5 times tailing reaction buffer (concentrated reaction buffer is

usually supplied from the manufacturer with the terminal transferase), 5 nmol

digoxigenin-11-dUTP (Boehringer Mannheim), 2 nmol dCTP (Pharmacia), and

30-50 units terminal deoxynucleotidyl transferase (Gibco BRL). The volume

was made up to 50 l with sterile deionised water.2. Reaction was incubated at 37C for upto 1 hour.

3. Reaction was stopped with addition of 10l EDTA (0.25M pH8) and placed

on ice.

4. Tailed probe was recovered by adding; 10g tRNA, 6l 3M sodium acetate

(pH 6.0) and 150l ice cold ethanol, to the reaction mix and incubating at -20C

for at least 2 hours. The DNA was then pelleted by centrifugation at 120000g in

a micro-centrifuge for 10 min. After carefully removing the supernatant the

pellet was dried under vacuum and re-dissolved in 50l of sterile deionised

water and stored at -20C.

Pre-Hybridisation Treatment of Tissue Section

1. Paraffin wax embedded sections were heat fixed onto 3-

aminopropyltriethoxysilane coated slides.

2. Sections were de-waxed in xylene (2 x 15 min), rehydrated through 100%

and 70% ethanol and finally washed in deionised water.


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3. Pre-treatments included:

i) Hydrolysis in 0.2M HCl for 10 min.

ii) 0.3% Triton X-100 in l times TBS (0.1M Tris, 0.15M NaCl, pH8.2) for 15 min.

iii) Digestion with proteinase K (Sigma) in 0.lM Tris, 50mM EDTA, pH 8 for 30

min at 37C. (NB. Proteinase concentration is crit ical and a titration experiment

is necessary for each particular tissue block. Usually between 0-20g/ml is

optimal).

iv) 0.2% glycine for 1 min.

v) Post-fixation in 4% paraformaldehyde for 5 min.

vi) 0.25% acetic anhydride in 0.1M triethanolamine (pH 7.5) for 10 min.

(NB. Sections were washed briefly in l times TBS between each pre-treatment).

4. Sections were placed in pre-hybridisation buffer for 1 hour.

Item Volume l in 1ml

De-ionised Formamide1 500

100x Denhardts2 10

20x SSC3 100

Denatured Calf Thymus DNA (10 mg/ml) 25

Dextran Sulphate 200

SDS (10%) 50

Tris (0.5M, pH7.5) 20

De-ionised Sterile Water 95

Hybridisation

1. Excess pre-hybridisation buffer was carefully removed from around sectionand 20 l of pre-hybridisation buffer, containing between 10-100 ng (0.5-5g/ml) of digoxigenin labelled oligonucleotide, was applied.

2. Hybridisation solution was covered with a small square of paraffin film andincubated in a sealed humid container (Terasaki plate) at 42C for 18 hours.

3. Paraffin film was removed by soaking in 2x SSC.

4. Post-hybridisation washes to remove non-specifically bound probe DNAincluded: two changes of 2x SSC, 10 min each at 42C followed by twochanges of 0.1x SSC, 20 min each at 42C.

5. Finally sections were equilibrated in 1x TBS.


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Detection of Hybrids with Gold Labelled Antibody

1. After post-hybridisation washes sections were blocked with 1% casein in lxTBS containing 0.1% Tween 20 for 1 hour at 20C.

2. Blocking solution was shaken off and Sheep anti-Digoxigenin goldconjugate, diluted 1/100 in 0.1-0.5% casein, lx TBS, 0.1% Tween 20 wasapplied to the section and incubated for at least 2 hours.

3. Sections were washed thoroughly in lx TBS to remove excess antibody andfinally rinsed 5x 1min in deionised water.

4. Gold particles were visualised using silver enhancer, for approximately 20min or until the sections appeared optimally developed under light microscopecontrol. The reaction was stopped by rinsing in water for 1 min.


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In-situ Hybridisation 3

Some Practical Hints into the Technique of in-situ Hybridisation

in-situ hybridisation (ISH) is the only technique that enables the morphological

demonstration of specific DNA and RNA sequences in individual cells of tissue

sections and cytospins and in chromosomal preparations. The development of

non-isotopic DNA labelling techniques has enabled ISH to become a routine

procedure in many research and clinical laboratories.

However, although optimal conditions have been developed for DNA and RNA

hybridisation on solid supports, the use of tissue sections or cell preparations

present additional problems, such as non-specific binding, sensitivity of signal

detection and target accessibility. Here we describe some useful tips that are

not always apparent in scientific publications.

Contents

1. Tissue Treatment

2. Slide Handling

3. Tissue Permeabilisation

4. Background

5. Controls for Hybridisation

6. Probe Concentration

7. The use of Gold Conjugates for in-situ Hybridisation

1. Tissue Treatment

Fixatives

Fixation is aimed at preserving cell morphology and the nucleic acid, especially

RNA sequences, composition of tissue whilst allowing adequate probe

penetration during hybridisation. The aldehyde fixatives, paraformaldehyde and

glutaraldehyde, promote cross linking of proteins and provide better RNAretention and morphology than the precipitating fixatives such as Bouin's and

Carnoy's fluid. We have found that the use of Bouin's fixative greatly reduced

the level of RNA detection in sections.

Fixation in 4% paraformaldehyde is a popular compromise between

permeability and RNA retention. Make solution by dissolving 4g of

paraformaldehyde in 100ml of PBS by warming the mixture to 60C for 30 min

in a fume hood, (NB. Paraformaldehyde is a carcinogen). Allow solution to cool

and filter through Whatman #l paper before use. Always make fresh solution on


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same day. Tissue is usually fixed for up to 18 hours by immersion.

2. Slide Handling

Slide Coating

It is now widely accepted that the most efficient and convenient method ofadhering tissue sections to glass slides is the use of 3-

aminopropyltriethoxysilane (APS).

(a) Clean slides are dipped in a solution of 2% APS in acetone for 5 min.

(b) Immerse slides in deionised water for 2 x 5 min.

(c) Leave to air dry or warm at 37C. When completely dry store in boxes at

room temperature.

Slide Incubations

During the hybridisation process it is crucial that at no stage should the tissue

section be allowed to dry out We have found that two slides placed in a

Terasaki plate containing a small volume of water and placed in a humid

container (sandwich box) efficiently retains the hybridisation volume during the

overnight incubations at temperatures up to 42C.

The sections were also covered with a small square of paraffin film (just large

enough to cover section). Care was taken to avoid air bubbles. This procedure

avoids the tedious process of sealing coverslips with rubber solution.

When denaturation of target DNA was necessary the same apparatus was

used, the Terasaki plate containing the slides was placed on a preheated metal

plate in an oven at 95C for 10 minutes.

3. Tissue Permeabilisation

Permeabilisation is necessary to enable accessibility of the probe target

DNA/mRNA. This stage of ISH is critical to obtaining maximum signal to

background ratio and must be optimised for each particular tissue block or cellpreparation. For instance, heavily cross-linked tissue will require extensive

permeabilisation.

This treatment typically involves exposure to dilute acids, detergents, alcohols,

and proteases such as proteinase K, pronase, or pepsin. A common

permeabilisation procedure would include;

(a) Hydrolysis in 0.2 M HCl for 10 min.

(b) 0.3% Triton X-l00 in 1 x TBS (0.lM Tris, 0.15M NaCl, pH 8.2) for 15 min.


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(c) Digestion with proteinase K in 0.1M Tris, 50mM EDTA, pH8.0 for 30 min at

37C. This step is crit ical and a titration experiment is necessary to determine

the optimal amount of proteinase required, too little results in a poor signal and

too much can result in high background and poor tissue morphology. Usually

somewhere between 1-20 g/ml is appropriate.

(d) Retention of tissue morphology in the subsequent hybridisation procedure

is maintained by a post-fixation in 4% paraformaldehyde for 5 min.

4.Background

Reduction of background during hybridisation.

Non-specific background is mainly due to retention of probe in tissue sections

by physical entrapment and electrostatic actions among charged groups. To

decrease tissue "stickiness", a pre-hybridisation incubation was performedwhich was intended to saturate sites in the tissue that might otherwise bind

nucleic acids un-specifically. Pre-hybridisation buffer contains all the

components of the hybridisation buffer except the probe DNA. Pre-

hybridisation was usually performed for 1 hour at 37C, the solution was then

drained off and hybridisation buffer was applied directly onto the moist section.

Electrostatic attraction between probe and basic proteins can be reduced by

pre-treatment of sections with 0.25% Acetic Anhydride in 0.lM Triethanolamine

(pH 7.5) for 5 min, which blocks basic groups by acetylation.

Reduction of background during non-isotopic detection

The majority of non-isotopic detection systems rely on the use of a conjugated

antibody specific to a hapten labelled DNA/RNA probe. We have obtained our

best results using digoxigenin labelled DNA and detecting hybrids using a

Sheep anti-Digoxigenin gold conjugated antibody.

Non-specific antibody binding results from attraction to charged groups within

the section. Unfortunately these groups are further revealed by the proteinase

treatment prior to hybridisation. Also certain tissues types and even areas

within tissues are more prone to non-specific by charge attraction such asconnective tissue, cell membranes and nuclear proteins.

We found the most effective blocking regime to be incubation in 0.5-1% casein

in 1 x TBS containing 0.1% Tween 20 for 1 hour. The antibody was diluted in

0.1% casein, 1 x TBS, 0.1% Tween 20 and applied to section for 2 hours.

(Casein forms negatively charged micelles, which effectively saturate positively

charged groups in the tissue).

When a double layer antibody detection step was being used, the washes

between the primary and secondary antibodies were performed with solutions


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containing blocking reagents.

5. Controls for Hybridisation

The possibility of non-specific probe and antibody attraction emphasizes the

need for adequate controls to be performed. Some useful controls would

include:

1. Sections processed as normal but in the absence of labelled probe.

2. Sections hybridised with a non-specific or irrelevant labelled probe, e.g.

vector DNA or sense oligonucleotide probe.

3. During RNA detection one section can be subjected to RNAse A treatment

(100-200 g/ml in 2 x SSC for 2 hours at 37C) before hybridisation (N.B. it is

important that RNAse does not contaminate any of the incubations).

4. An initial hybridisation can be carried out with unlabelled probe in excess

followed by incubation with labelled probe.

6. Probe Concentration

Optimal probe concentration is difficult to predict however it is best to use the

lowest concentration required to saturate target nucleic acids. In our

experience the concentration for multi-primed or nick translated DNA probes

ranges from 0.1-0.5g/ml and for end labelled oligonuleotide between 0.5-

5.0g/ml. Preliminary experiments are necessary to determine the optimal

signal to noise ratio.

Estimation of oligonucleotide DNA concentrationA typical 0.25 mole synthesis will yield between 50 and 150 nmol of full-length

oligonucleotide. Oligonucleotide concentration is usually reported in

absorbance units at 260nm that can be converted into molar units by assumingthat 1 A260 unit = 33 g of oligonucleotide.

For example, a 24 mer oligonucleotide has an A260 of 0.2 when diluted 1/200.

x 33 g x 200 (dilution factor) = 1.3 mg/ml

- (oligo length x 330 g/mol) = 0.16 mol

x 1,000 nmol/mol = 160 nmol

Before using any labelled probe an estimate of its sensitivity should be made

using a dot blot hybridisation technique. This involves blotting dilutions of

target DNA or RNA onto a membrane and hybridising with a known

concentration of probe. Sometimes target DNA is not readily available and a

direct blot of the probe can be made to assess efficiency of labelling.


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7. The use of Gold Conjugates for in-situ Hybridisation

Gold conjugated antibodies have been used extensively for immunohistological

labelling of cells and tissue sections. Now gold conjugates, along with enzyme

and fluorescent labelled antibodies, in combination with non-radioactively

labelled DNA and RNA probes are rapidly replacing the use of radioactively

labelled probes for in-situ hybridisation. Gold conjugates provide a number of

important benefits to the technique of in-situ hybridisation.1. A very distinct signal is obtained. This is viewed as an intense black reaction

using bright field microscopy or as a brilliant reflective image under

epipolarised illumination. Other labels, such as enzymes, can result in diffuse

signals that may be misleading.

2. Silver enhancement is rapid. Maximum sensitivity is obtained after 20-30

min.

3. The signal is enhanced further (approximately 10 fold) using epipolarizing

illumination. This also provides a spectacular light microscope image of the

labelled section.

4. The signal is insoluble in most laboratory dehydrating and mounting agents,

does not fade with time and is compatible with a wide variety of counter stains.

5. There is no interference from endogenous enzyme activity.

6. Hybridisation studies can be further investigated at the electron microscope

level. Various sizes of gold particles are available for multiple labelling.

7. Individual gold particles can be counted in the electron microscope to give a

quantitative estimation of gene expression or gene copy number. It is also

possible to count individual gold/silver enhanced particles using high power

magnification in the light microscope.

Publications

In-situ HYBRIDISATION - Principles and practice

PolakJM and McGee J (1992)

DNA PROBES

Keller G and Manak M (1992)

In-situ HYBRIDISATION A practical approachDG Wilkinson (1995)

In-situ HYBRIDISATION A practical guide

Leitch AR et al (1994)

Documents

In-Situ Hybridisation