C. SUMMARY
D. Selected Bibliography-In Situ Hybridization Protocol
E. References
F. Protocols and Reagents

1. In Situ of Frozen Sections Using 35S Riboprobes
2. 35S-Riboprobe Synthesis
3. Labeling of Synthetic Oligomers with 35S-CTP
4. Tissue Preparation Protocol
5. Buffers and Solutions of In Situ

In situ hybridization provides the researcher invaluable information regarding the localization of gene expression in heterogeneous tissues. The technique is extremely sensitive and can detect the amount of mRNA contained in a single cell. However be careful how you apply this technique. In situ hybridization looks good and is often used not because of scientific necessity but rather for the flash it adds to a presentation. In situ is a difficult procedure and one has to ask whether it is worthwhile to develop it for a single experiment, or if a Northern blot or some other assay might provide the necessary information instead with a lot less effort.

I have tried to provide a starting point for those individuals who wish to begin using in situ hybridization in their own laboratories. The procedure as outlined in this manuscript has been used with many probes and tissues with a greater than 90% success rate on the first hybridization. This protocol was designed to be compatible with tissues coming from many different sources including surgical biopsy, autopsy, and animal experimentation. The procedure is streamlined with the goal of being able to hybridize as many as 100-200 slides per day with at least four different probes. Many steps such as acetylation, defatting in xylene or alcohols, post-fixation, or multiple proteinase steps have been eliminated. These are often used to reduce backgrounds in the tissue. However if this procedure is applied correctly backgrounds are not a problem. A good hybridization will have 25-50 cytoplasmic (not nuclear) grains per positive cell and less than 1 grain per negative cell or cell sized area of tissue matrix. The improvement in the signal to noise ratio comes from the use of ribonuclease and high stringency washes in the post-hybridization steps. While these washes do reduce the specific signal they enhance the ultimate level of detection of low copy number mRNAs by increasing the signal to noise ratio.

This protocol is a good starting point for those who wish to use in situ hybridization in their work. While it is possible that the hybridization signal can be improved in individual situations, do not make changes before you start. The best recommendation is to use it exactly as stated until favorable results are obtained and once there is a signal to work with then begin to make controlled changes in the procedure and see what effect they have, if any, on the signal intensity.

Be cautious about controls and what is accepted as a positive hybridization signal. It is very easy to get silver grains on the tissue section after hybridization and call it positive. For example regions of the tissue rich in nuclei often appear to cause spurious binding of the probe and have high backgrounds. The trick is not to get clusters of silver grains on the slide but rather to do the appropriate controlled experiments to assure yourself that the signal is real and not due to some artifactual binding of the probe to the tissue. Most of all use common sense in interpreting your results and challenge yourself by performing simultaneous positive and negative controls to be certain of the specificity of the hybridization.

1. Wilcox, J.N. Fundamental principles of in situ hybridization. J. Histochem. Cytochem. 41, 1725-1733, 1993.

2. Wilcox, J.N., Smith, K.S., Williams, L.T., Schwartz, S., and Gordon, D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J. Clin. Invest. 82, 1134-1143, 1988.

3. Wilcox, J.N., Smith, K.M., Schwartz, S.M., Gordon, D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl. Acad.Sci. 86, 2839-2843, 1989.

Riboprobe transcription - Melton, D.A., Krieg, P.A., Rebagliati, M.R., and Maniatis, T., Zinn, K., Green, M.R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12, 7035-7056, 1984.

First description of the use of riboprobes for in situ hybridization - Cox, K.H., DeLeon, D.V., Angerer, L.M., and Angerer, R.C. Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485-502, 1984.

Overview of in situ hybridization - Wilcox, J.N., Gee, C.E., and Roberts, J.L. In situ cDNA:mRNA hybridization: Development of a technique to measure mRNA levels in individual cells. In: Methods in Enzymology, Vol 124, Neuroendocrine Peptides (P.M. Conn, ed.), Academic Press, pp510-533, 1986.

Trancriptional analysis using in situ hybridization of intron specific probes - Fremeau, R.T., Lundblad, J.R., Pritchett, D.B., Wilcox, J.N., and Roberts, J.L. Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei. Science 234, 1265-1269, 1986.

Comparison of Northern blots, film autoradiography, and nuclear emulsion localization - Rosenthal, A., Chan, S.Y., Henzel, W., Haskell, C., Kuang, W-J., Chen, E., Wilcox, J.N., Ullrich, A., Goedel, D.V., and Routtenberg, A. Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate, associated with synaptic plasticity. EMBO J. 6,3641-3646, 1987.

In situ hybridization of mouse embryos - Wilcox, J.N. and Derynck, R. Developmental expression of transforming growth factors alpha and beta in mouse fetus. Molec. Cell. Biol. 8,3415-3422, 1988.

In situ localization in the brain - Wilcox, J.N., Pollard, H. Moreau, J., Schwartz, J.C., Malfroy, B. Localization of enkephalinase mRNA in rat brain by in situ hybridization: Comparison with immunohistochemical localization of the protein. Neuropeptides 14, 77-83, 1989.

Combined in situ/axonal tracing technique - Wilcox, J.N., Roberts, J.L., Chronwall, B.M., Bishop, J.F., and O'Donohue, T. Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections. J. Neurosci. Res. 16, 89-96, 1986.

Angerer LM, Cox KH, Angerer RC, Demonstration of tissue-specific gene expression by in situ hybridization, Methods Enzymol, 152, 649-661, 1987.

Armstrong E, Partanen J, Cannizzaro L, Huebner K, Alitalo K, Localization of the fibroblast growth factor receptor-4 gene to chromosome region 5q33-qter, Genes Chromosom Cancer, 4, 94-98, 1992.

Brahic M, Haase AT, Detection of viral sequences of low reiteration frequency by in situ hybridization, Proc Natl Acad Sci U S A, 75, 6125-6129, 1978.

Chen RH, Fuggle SV, In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression, American Journal of Pathology, 143, 1527-1534, 1993.

Conkie D, Affara N, Harrison PR, Paul J, Jones K, In situ localization of globin messenger RNA formation. II. After treatment of Friend virus-transformed mouse cells with dimethyl sulfoxide, J Cell Biol, 63, 414-419, 1974.

Cox KH, DeLeon DV, Angerer LM, Angerer RC, Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes, Dev Biol, 101, 485-502, 1984a.

Cox KH, DeLeon DV, Angerer LM, Angerer RC, Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes, Dev Biol, 101, 485-502, 1984b.

Fremeau RT, Lundblad JR, Pritchett DB, Wilcox JN, Roberts JL, Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei, Science, 234, 1265-1269, 1986.

Fremeau RT, Autelitano DJ, Blum M, Wilcox J, Roberts JL, Intervening sequence-specific in situ hybridization: detection of the pro-opiomelanocortin gene primary transcript in individual neurons, Brain Res Mol Brain Res, 6, 197-201, 1989.

Gall JG, Pardue ML, Formation and detection of RNA-DNA hybrid molecules in cytological preparations, Proc Natl Acad Sci U S A, 63, 378-383, 1969.

Gee CE, Chen CL, Roberts JL, Thompson R, Watson SJ, Identification of proopiomelanocortin neurones in rat hypothalamus by in situ cDNA-mRNA hybridization, Nature, 306, 374-376, 1983.

Gee CE, Roberts JL, In situ hybridization histochemistry: a technique for the study of gene expression in single cells, DNA, 2, 157-163, 1983.

Gordon D, Augustine AJ, Smith KM, Schwartz SM, Wilcox JN, Localization of cells expressing tPA, PAI1, and urokinase by in situ hybridization in human atherosclerotic plaques and in the normal rhesus monkey, Thromb Haemost, 62, 131, 1989.(abstract)

Harrison PR, Conkie D, Paul J, Jones K, Localisation of cellular globin messenger RNA by in situ hybridisation to complementary DNA, FEBS Letters, 32, 109-112, 1973.

Heniford BW, Shum-Siu A, Leonberger M, Hendler FJ, Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction, Nucleic Acids Res, 21, 3159-3166, 1993.

Hudson P, Penschow J, Shine J, Ryan G, Niall H, Coghlan J, Hybridization histochemistry: use of recombinant DNA as a "homing probe" for tissue localization of specific mRNA populations, Endocrinology, 108, 353-356, 1981.

Komminoth P, Long AA, In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique, Virchows Arch B Cell Pathol Incl Mol Pathol, 64, 67-73, 1993.

Lawrence JB, Singer RH, Quantitative analysis of in situ hybridization methods for the detection of actin gene expression, Nucleic Acids Res, 13, 1777-1799, 1985.

Leclerc G, Isner JM, Kearney M, Simons M, Safian RD, Baim DS, Weir L, Evidence implicating nonmuscle myosin in restenosis. Use of in situ hybridization to analyze human vascular lesions obtained by directional atherectomy, Circulation, 85, 543-553, 1992.

Majesky MW, Reidy MA, Bowen Pope DF, Hart CE, Wilcox JN, Schwartz SM, PDGF ligand and receptor gene expression during repair of arterial injury, Journal of Cell Biology, 111, 2149-2158, 1990.

Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR, Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nucleic Acids Res, 12, 7035-7056, 1984.

Nelken NA, Coughlin SR, Gordon D, Wilcox JN, Monocyte chemoattractant protein-1 in human atheromatous plaques, Journal of Clinical Investigation, 88, 1121-1127, 1991.

Nelken NA, Soifer SJ, OKeefe J, Vu TK, Charo IF, Coughlin SR, Thrombin receptor expression in normal and atherosclerotic human arteries, Journal of Clinical Investigation, 90, 1614-1621, 1992.

Nikol S, Isner JM, Pickering JG, Kearney M, Leclerc G, Weir L, Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions, Journal of Clinical Investigation, 90, 1582-1592, 1992.

Nuovo GJ, Darfler MM, Impraim CC, Bromley SE, Occurrence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction, American Journal of Pathology, 138, 53-58, 1991a.

Nuovo GJ, Gallery F, Mac Connell P, Becker J, Bloch W, An improved technique for the in situ detection of DNA after polymerase chain reaction amplification, American Journal of Pathology, 139, 1239-1244, 1991b.

Patel VG, Shum-Siu A, Heniford BW, Wieman TJ, Hendler FJ, Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction, American Journal of Pathology, 144, 7-14, 1994.

Pochet R, Brocas H, Vassart G, Toubeau G, Seo H, Refetoff S, Dumont JE, Pasteels JL, Radioautographic localization of prolactin messenger RNA on histological sections by in situ hybridization, Brain Res, 211, 433-438, 1981.

Rosenthal A, Chan SY, Henzel W, Haskell C, Kuang WJ, Chen E, Wilcox JN, Ullrich A, Goeddel DV, Routtenberg A, Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity, EMBO J, 6, 3641-3646, 1987.

Simons M, Leclerc G, Safian RD, Isner JM, Weir L, Baim DS, Relation between activated smooth-muscle cells in coronary-artery lesions and restenosis after atherectomy, N Engl J Med, 328, 608-613, 1993.

Wilcox JN, Gee CE, Roberts JL, In situ cDNA:mRNA hybridization: development of a technique to measure mRNA levels in individual cells, Methods Enzymol, 124, 510-533, 1986.

Wilcox JN, Derynck R, Developmental expression of transforming growth factors alpha and beta in mouse fetus, Mol Cell Biol, 8, 3415-3422, 1988.

Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D, Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization, Journal of Clinical Investigation, 82, 1134-1143, 1988.

Wilcox JN, Pollard H, Moreau J, Schwartz JC, Malfroy B, Localization of enkephalinase mRNA in rat brain by in situ hybridization: comparison with immunohistochemical localization of the protein, Neuropeptides, 14, 77-83, 1989a.

Wilcox JN, Smith KM, Schwartz SM, Gordon D, Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque, Proc Natl Acad Sci U S A, 86, 2839-2843, 1989b.

Wilcox JN, Analysis of local gene expression in human atherosclerotic plaques, J Vasc Surg, 15, 913-916, 1992.

1. Combine:

1.6µl sdH2O

2.0µl DNA (approx 10ng/µl stock)

2.0µl 5x Terminal Transferase buffer (BRL will provide with enzyme)

3.4µl 35S-labelled dCTP (50pmol)

1.0µl TdT enzyme (BRL, approx 14 units)

_________

10.0µl Total

2. Incubate 45min at 37°C

3. Place on ice, spot 1µl on DE81 paper to determine specific incorporation

(see riboprobe procedures for DE81 method)

4. Add 20µl 1xTE (30µl total), add 15µl phenol, vortex, add 15µl cloroform, vortex, centrifuge for 3min, remove aqueous top phase to clean tube (30µl); reextract the organic phase with another 15µl 1xTE, vortex, centrifuge, add the top phase to the previous extract (45µl total).

5. Separate free nucleotides from sample using Biogel P6 columns or any commercial columns for oligo purification (ie. Boehringer Mannheim G-25 Quick Spin columns). Do not purify using ethanol precipitation as there may be loss of the oligo due to its size.

Note: It is critical that the 35S-labeled CTP be supplied as an aqueous solution without DTT. DTT in excess of 1mM will cause the transcription buffer components to precipitate out and inhibit the reaction.

1. Remove tissue and rinse in PBS or saline

2. Immerse in 4% paraformaldehyde 0.1M sodium phosphate buffer pH7.4 at 4°C for 1-3hrs. Try to avoid overnight fixation if possible as this causes problems with keeping the section on the slide during the hybridization procedure.

3. Immerse in sterile 15% sucrose-PBS solution 3hrs-overnight at 4°C.

4. Embed tissue in °.C.T. (Baxter #M7148-4), M1 (Lipshaw) or any other convenient embedding matrix for frozen sectioning. Tissue should be oriented in the block appropriately for sectioning (cross-section etc.). Note the tissue number on the block directly and indicate which face of the block should be sectioned.

5. Freeze block with tissue in liquid nitrogen. Place approximately the bottom third of the block into the liquid nitrogen, allow to freeze until all but the center is frozen, allow freezing to continue on dry ice.

6. Store at -70°C in a sealed container or wrapped in foil and ship on dry ice.

It is also possible to use fresh frozen tissue for in situ hybridization if the paraformaldehyde/sucrose method is not feasible. Tissue should be rinsed in saline or PBS and frozen in liquid nitrogen in O.C.T. blocks as outlined above. Although not optimal it is also possible to use snap frozen material tissue without an embedding matrix. The fixation, sucrose, and O.C.T. steps are used primarily to improve the tissue morphology.

It is expected that the fixation times outlined above will not result in complete fixation of large pieces of tissue. However, the fixation step at the beginning of the hybridization procedure should ensure adequate fixation of such tissues prior to hybridization.

This protocol has been used successfully on large (up to 1cm3) and small (1mm3) tissue samples.

Use 1.2mm thick slides as the 1.0mm slides tend to break more easily. Load slides into metal holder, 50 slides per rack. Wash as follows:

0.2M NaOH, 30min, RT.

tap distilled H2O 10min, RT.

Glass distilled H2O 2min, RT.

Ethanol (reagent grade) 2min, RT.

Ethanol (reagent grade) 2min, RT.

Dry in oven at 42°C, then dip in:

100µg/ml poly-l-lysine in dH20 for 20 seconds.

(Poly-L-lysine MW 47,000, Sigma cat# P-2636)

Allow slides to dry for 30 minutes at RT, then redip in same poly-L-lysine solution. Dry in oven overnight at 42°C. Store for up to six months at RT.

We have found an improved method of adhering tissue to slides for in situ hybridzation that uses a prepared reagent VectaBond (Cat #SP1800 - Vector Labs, Burlingame, Ca.). Slides are coated essentially following manufacturer's directions.

1. Dip 5min in acetone.

2. Place slides in Vectabond/acetone (1 bottle/350mls) for 5min.

3. Rinse in dH2O for ~1min with some agitation.

4. Drain on clean diaper.

5. Dry overnight at 42°C.

6. Put slides back in original boxes and store indefinately at room temp.

We have recently begun to use SuperFrost/Plus microscope slides for all of our frozen tissue sectioning and have very good results as regards tissue retention on the slide after in situ hybridization. All three methods of slide coating appear to have similar properties as regards tissue retention. The advantage of using SuperFrost/Plus slides is they require no preparation time in the laboratory and are competitive in terms of cost when you consider technician time and reagent expenses.

Frozen tissues prepared as described can be wrapped and stored for many years prior to sectioning without loss of the mRNA signal. The biggest problem with stored tissue blocks is that they tend to dessicate if not wrapped properly and the OCT can be difficult to cut.

Blocks should be removed from the -70°C freezer and allowed to equilabrate with the cryostat chamber temperature. Tissues can be cut at any convenient temperature (-15 to -35°C) as needed. Most tissues cut well at -15°C (brain, kidney, liver, vessels, muscle, etc.) however fatty or more difficult tissues (adipose tissue, skin, lung) require temperatures as low as -35°C or more to obtain good sections. Vectabond coated slides should be kept at room temperature. Care should be taken not to touch the face of the slides but handle by the edges only. Frozen sections 5-7µm (thinner is OK but thicker, over 10µm, may present problems) should be cut, thaw-mounted on the room-temperature coated slides, and the slide with the section immediately refrozen by placing into a slide box (VWR micro slide box #48444-003) with a single dessicant capsule (Humi-Cap see below). When the box is full, place the top on the box and store at -70°C. Sections cut and stored with dessicant are stable for in situ hybridization and immunohistochemistry for most antigens for over 5 years.

rHB2 Hybridization Buffer (for riboprobes)

	Stock Concentration	Volume of Stock
10mM DTT	100%	46.26mg
sdH2O		5.7ml
0.3M NaCl	5M	1.8ml
20mM TRIS, pH8.0	1M	600µl
5mM EDTA	250mM	600µl
1x Denhardt's	100x	300µl
10% Dextran Sulfate	50%	6.0ml
50% Formamide	100%	15.0ml
Total Volume		30.0ml

HB8 Hybridization Buffer (for oligos)

	Stock Concentration	Volume of Stock
10mM DTT	100%	46.26mg
sdH20		9.84ml
1x Denhardt's	100x	300µl
5xSSC	20x	7.5ml
100µg/ml ssDNA	10mg/ml	300µl
100µg/ml tRNA	50mg/ml	60µl
10% Dextran Sulfate	50%	6.0ml
20% Formamide	100%	6.0ml
Total Volume		30.0ml

RNAse Buffer

	Stock Concentration	Volume of Stock
500mM NaCl	5M	100ml
10mM TRIS, pH8.0	1M	10ml
dH20		890ml
Total Volume		1000ml

RNAse Stock (10mg/ml)

10mg RNAse A (Sigma)

1.0ml RNAse Buffer

Heat treat as per Maniatis 1st edition p.451

Working RNAse Solution-20mg/ml

300µl RNAse Stock in 150ml RNAse Buffer

2xSSC, bME, EDTA

	Stock Concentration	Volume of Stock
2x SSC	20x	100ml
10mM beta-mercaptoethanol	100%	875µl
1mM EDTA	250mM	4.0ml
dH20		860ml
Total Volume		1000ml

Box Buffer

	Stock Concentration	Volume of Stock
4x SSC	20x	50ml
50% Formamide	100%	125ml
dH20		75ml
Total Volume		250ml

Stringency Buffer

	Stock Concentration	Volume of Stock
0.1xSSC	20xSSC	20ml
10mM	beta-mercaptoethanol	3.5ml
1mM EDTA	250mM	16.0ml
dH20		3960.5ml
Total Volume		4000ml

Dehydration Buffers:

	50%	70%	90%	100%
100% EtOH	100ml	140ml	180ml	200ml
3M NH4Ac	20ml	20ml	20ml	--
dH20	80ml	40ml	--	--
Total Volumes	200ml	200ml	200ml	200ml

50x Denhart's
1g Ficoll
1g Polyvinylpyrrolidone
1g BSA
Add sterile distilled H2O to 100ml

Proteinase K
(20ml/ml) stock made up in water, frozen in aliquots which are used only once

4% Paraformaldehyde

Mix in a two liter flask:

200ml 0.5M NaPO4, pH 7.4
800ml depcH20
Heat to 70°C with stirring on hot plate in fume hood
Add 40g Paraformaldehyde (EM grade, Polysciences, Cat No. 0380)

Once the solution has cleared (it should take 5 minutes or less), filter with a side-arm flask, Buchner funnel and Whatman No. 2 filter paper.
Immediately pour the solution into a one liter bottle which has been packed in ice. This cools the solution quickly and prevents breakdown of the paraformaldehyde. Store at 4°C for up to two weeks.

15% Sucrose in PBS:

500ml sterile PBS
75g "RNase free" sucrose

Mix above and filter sterilize with a disposable Nalgene filtration unit type S(0.45 micron). Store at 4°C.