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 DNA probes 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 3aminopropyltriethoxysilane 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 biotin (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

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 antiBiotin 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 Formamide1 100x Denhardts2 20x SSC3 10 mg/ml calf thymus DNA De-ionised water Probe DNA Volume l/ml 500 50 100 20 30 Upto 300 200 ng/ml Working 50% 5x 2x 200 g/ml

1. Formamide is a teratogen. Handle with extreme care and wear gloves. To deionise 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 filter paper to remove the resin. Store at 4C in a dark bottle.

2. 100x Denhardt's solution is 2% Ficoll (mw 400,000), 2% polyvinyl pyrrolidone (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 by autoclaving.

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 3aminopropyltriethoxysilane 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.

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 critical 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 De-ionised Formamide1 100x Denhardts2 20x SSC3 Denatured Calf Thymus DNA (10 mg/ml) Dextran Sulphate SDS (10%) Tris (0.5M, pH7.5) De-ionised Sterile Water Volume l in 1ml 500 10 100 25 200 50 20 95

Hybridisation
1. Excess pre-hybridisation buffer was carefully removed from around section and 20 l of pre-hybridisation buffer, containing between 10-100 ng (0.5-5 g/ml) of digoxigenin labelled oligonucleotide, was applied. 2. Hybridisation solution was covered with a small square of paraffin film and incubated 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 DNA included: two changes of 2x SSC, 10 min each at 42C followed by two changes of 0.1x SSC, 20 min each at 42C. 5. Finally sections were equilibrated in 1x TBS.

Detection of Hybrids with Gold Labelled Antibody

1. After post-hybridisation washes sections were blocked with 1% casein in lx TBS containing 0.1% Tween 20 for 1 hour at 20C. 2. Blocking solution was shaken off and Sheep anti-Digoxigenin gold conjugate, diluted 1/100 in 0.1-0.5% casein, lx TBS, 0.1% Tween 20 was applied to the section and incubated for at least 2 hours. 3. Sections were washed thoroughly in lx TBS to remove excess antibody and finally rinsed 5x 1min in deionised water. 4. Gold particles were visualised using silver enhancer, for approximately 20 min or until the sections appeared optimally developed under light microscope control. The reaction was stopped by rinsing in water for 1 min.

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 RNA retention 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

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 of adhering tissue sections to glass slides is the use of 3aminopropyltriethoxysilane (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 cell preparation. 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.

(c) Digestion with proteinase K in 0.1M Tris, 50mM EDTA, pH8.0 for 30 min at 37C. This step is critical 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 performed which 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. Prehybridisation 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 as connective 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

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 0.55.0g/ml. Preliminary experiments are necessary to determine the optimal signal to noise ratio.

Estimation of oligonucleotide DNA concentration

A 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 assuming that 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.

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 Polak JM and McGee J (1992) DNA PROBES Keller G and Manak M (1992) In-situ HYBRIDISATION A practical approach DG Wilkinson (1995) In-situ HYBRIDISATION A practical guide Leitch AR et al (1994)