In situ hybridization for localization of m RNAs in mononuclear phagocytes in cell culture and tissue sections

JOURNALOF IMMUIlOLOGICAL METHOI)S Journal of Immunological Methods 174 (1994) 281-296

ELSEVIER

In situ hybridization for localization of mRNAs in mononuclear phagocytes in cell culture and tissue sections Jean Michel Vignaud

*, N a d i n e

Martinet,

Yves Martinet,

Francois Plenat

Laboratoire d'Anatomie Pathologique, INSERM Unitd 14, Service de Pneumologie, CHU, Nancy, France

Abstract

We report an in situ hybridization procedure to detect in cell preparations and tissue sections messenger RNAs coding for mononuclear phagocyte proteins. The multistep procedure is described for use in conjunction with isotopic and non-isotopic probes. Keywords: In situ hybridization; Monocyte; Macrophage; Messenger RNA

1. Introduction

In situ hybridization is a very sensitive and specific technique used to determine, with a high degree of spatial resolution, the cellular a n d / o r tissular localization of D N A and R N A sequences in cytological and histological preparations. The sensitivity of the method is such that a few copies of a specific m R N A can be detected within a single monocyte cell. In addition the method can provide quantitative information about m R N A content using a computer-assisted image analysis

Abbreviations:ISH, in situ hybridization; DEPC, diethylpyrocarbonate; PFA, paraformaldehyde; DTT, dithiothreitol; TdT, terminal deoxynucleotidyl transferase; BCIP, bromo chloro indolyl phosphate; NBT, nitro blue tetrazolium. * Corresponding author. At" Laboratoire d'Anatomie Pathologique, CHU de Nancy, H6pital Central, 29, av. du Marechal-de-Lattre-de-Tassigny, C.O. n° 34, 54035 Nancy Cedex, France. Tel.: (33)83.85.13.51; Fax: (33)83.85.11.49.

system. The aim of this article is to describe the radioactive and non-radioactive procedures used in our laboratory for the detection of m R N A s (Vignaud et al., 1991). The protocols described have worked well with a wide range of D N A and R N A probes and for different cells and tissues. The application to mononuclear phagocytes does not raise any specific problems, although some cell preparations or tissues may require slight modifications of the basic procedure to optimize the results. Indeed this powerful and versatile technique does not respond to a standard protocol. Furthermore it must be emphasised that the individual steps that constitute the final protocol are interdependent, and the modification of one of them can interfere with the others. For further dicussion of the method and alternate protocols the reader should consult the following reviews: H a m e s and Higgins (1985), Valentino et al. (1987), Heller and M a n a k (1989), Polak and M c G e e (1990), in conjunction with one of the guides to

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basic techniques in molecular biology such as: Molecular Cloning - A Laboratory Manual (Sambrook et al., 1989), A Practical Guide to Molecular Cloning (Perbal, 1988), or Current Protocols in Molecular Biology (Ausabel et al., 1990), that provide a number of practical details. The multistep technique will be described in four sections as follows: cell and tissue preparation, probe labelling, hybridization, and signal detection.

2. Working in an RNase-free environment

In order to detect mRNAs successfully it is necessary to provide an environment which is RNase-free throughout the procedure. To avoid RNase contamination of samples and incubation solutions, disposable gloves must be worn and both instruments and glassware should be baked at 250°C for 5 h. Distilled water and all buffers (Tris buffers excepted) should be D E P C treated. Add 0.04% D E P C (v/v) to the water and buffers, stir vigorously until dissolution, incubate at 37°C overnight. D E P C inactivates RNases by covalent modification of primary amines or imidazole nitrogens on histidine. The reactivity of D E P C towards amines (e.g., in nucleic acids) dictates that D E P C should be destroyed by autoclaving (120°C, 30 min) the solutions before use.

3. Cell and tissue preparation

Monocytes and macrophages can be investigated from tissues either embedded in paraffin and sectioned on a microtome, or frozen and sectioned on a cryostat and from cells (primary sample or culture) deposited on slides by cytocentrifugation or grown on glass chamber slides. The critical point of this step is to fix samples as soon as possible once harvested and attach them to properly primed slides. 3.1. Microscope slide preparation Samples undergoing relatively harsh treatments during the ISH technique must be mounted

in a manner that will prevent their detachment from the slides. Silanisation of slides after hydrofluoric acid pretreatment gives the best result of the various methods proposed (Denhardt, polylysine or gelatine-chrome alum coated slides). The slides are prepared as follows. Carefully immerse clean frosted-ended slides, loaded into stainless steel racks, for 15 s in concentrated hydrofluoric acid. It is a very reactive acid: always wear gloves and goggles and work in a fume hood. Drain and wash the slides thoroughly for 10 min in running tap water. Dry the slides in a convection oven for 30 min at 120°C. Allow slides to cool at 50°C, then dip in 2% (v/v) 37aminopropyltriethoxysilane in acetone for 15 s. Dip in two changes of acetone for 15 s each, drain and let the slides dry overnight at 37°C. The slides are then stored at room temperature in a dry atmosphere and should be used within 6 months. 3.2. Fixation of cells and tissues It is essential to fix cells and tissues to retain the mRNAs and preserve a good morphology. Fixation must take place with the minimum delay possible because some mRNAs have a very short life (e.g. 30 min for c-rnyc). Furthermore, fixation time must be relatively brief to preserve the accessibility of the probe to targets and to prevent a high background. A panel of fixatives can be used either cross-linking or precipitating, depending on the tissue to be investigated and the probe size. The advantages and disadvantages of each fixative have been reviewed by Lawrence and Singer (1985), McAllister and Rock (1985) and Moench et al. (1985). Ethanol-acetic acid (3/1, v/v), is a very good fixative for the investigation of D N A sequences but a poor fixative for mRNAs because it has a weak capacity for the retention of RNAs. In contrast glutaraldehyde (2%) is a harsh cross-linking fixative producing abundant intra- and intermolecular bridging allowing high R N A retention but limiting the penetration of probes into cells. It is used with short probes (less than 150 nucleotides) and permeabilisation steps.

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Preserving mRNAs almost as well as glutaraldehyde but with less cross-linking, paraformaldehyde (4%) permits the utilisation of larger probes (400 nucleotides). It routinely gives us more reproducible results with regard to good RNA retention and target accessibility. Fixative solution: 4% PFA, 2% sucrose, 5 mM MgC12, 0.02% DEPC in 1 x PBS. Prepare fixative by dissolving sucrose and PFA at a concentration of 4% (w/v) in 1 X PBS. Heat to 60°C for approximately 30 min in a hood until solution clears. Cool at room temperature, add MgCI z and DEPC, adjust to pH 7.4, filter and store at 4°C (12 h maximum).

3.3. Cell preparation Plate cells on slides or let cells grow on chamber slides. Before fixation wash cells in 1 x PBS (2 changes of 5 min each) and fix in 4% PFA for 3 min. Or cytospin preparation: (1) suspend cells at 106/ml in culture medium; (2) load 250 ~1 cell suspension per slide; (3) spin at 800 rpm for 10 min in cytospin; (4) remove slides from machine holders; (5) let slides dry at room temperature for 15 min and fix in 4% PFA, for 5 min only. Wash fixed cells twice in 1 x PBS, use immediately or store either desiccated at - 70° C (use within one month) or in 70% ethanol at 4°C (6 months).

3.4. Frozen tissue section preparation Prepare a bath of isopentane in a polypropylene container dipped in liquid nitrogen. When white spots appear at the bottom of the container (is0pentane is cooled at - 160°C) place specimen in isopentane for 30 s and store frozen sample at - 70oc. Make cryostat sections 5-10 /zm thick and place on silanised slides. Dry sample at room temperature for 15 min and fix in 4% PFA for 5 min. Wash twice in 1 x PBS (5 min each change). Process sections immediately or store either desiccated at - 7 0 °C (use within one month) or in 70% ethanol (up to 6 months).

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3.5. Preparation of paraffin-embedded tissue sections Cut tissues into slices no more than 3-4 mm thick and fix them as soon as possible once harvested. Fix samples in 4% PFA, for 3-4 h at 4°C. Wash it in a large volume of 1 X PBS for 1 h (4°C) using two changes. Then place in 70% ethanol at 4°C until ready for paraffin infiltration. Tissues embedded in paraffin can be stored at 4°C for several months without significant loss of signal. Section cutting: place paraffin blocks on paraffin microtome and cut 8 /zm sections. Affix sections to primed slides by floating on a drop of DEPC-treated water. Place slides in an oven at 60°C for 10 min, transfer at 37°C for 1 h, and store at 4°C until use. Deparaffinize sections in xylene (2 changes, 10 min each) using a glass slide jar, remove xylene with absolute ethanol (2 changes, 5 min each), then in 70% ethanol (5 min), and rehydrate in 2 changes of 1 x PBS (5 min each). Specimens are now ready for hybridization. Deparaffinized sections can also be kept in 70% ethanol at 4°C for 6 months without significant loss of signal.

4. P r o b e l a b e l l i n g

cRNA, cDNA and oligodeoxynucleotide probes are suitable for in situ hybrydization. In practice the type of probe used will depend on what is available and on the experience of the laboratory in molecular cloning methods.

4.1. General considerations 4.1.1. Types of probes Riboprobes are the most sensitive tool for ISH. Single stranded, free of vector sequences, they give RNA-RNA hybrids of high stability allowing further stringent hybridization and post-hybridization washes. Furthermore in the post-hybridization step it is possible to use RNases to digest unhybridized probe (single stranded), resulting in

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a low background. Their synthesis requires suitable plasmids (e.g. pGEM, pBluescript) with a RNA polymerase promoter (SP6, T7 or T3) at either end of a polylinker where the cDNA of interest is inserted. These opposite distinct promoters allow the transcription of antisense (complementary) and sense (non-complementary, used as control) RNA probes. Double-stranded cDNA probes were the first to be used and are still widely utilized. They are less sensitive than riboprobes. Indeed RNA-DNA hybrids are less stable than RNA-RNA hybrids and the denatured double-stranded probe reanneals partially during hybridization. They are used with or without the vector sequences which may amplify the signal by networking (Lawrence and Singer, 1985). They are more convenient to handle than riboprobes, do not require subcloning of the sequences of interest and are easily labelled by nick translation or random primer extension. Synthetic oligodeoxynuclotide probes can be home made or purchased commercially, provided that the sequence of interest is published. These short probes ought to discriminate between related target sequences. Furthermore they have good cellular penetration. However, the hybrids are less stable than those built up with cDNA or cRNA probes and the hybridization conditions used are thus critical.

4.1.2. Choice of probe labelling Two main types of labelling strategy can be used: radioactive and non-isotopic. Problems of safety, reduced stability of radioactive probes, and speed of visualization have stimulated the development of non-isotopic probes. However, their sensitivity in routine use does not fully reach those of radioactive probes, in spite of constant improvements in the detection systems. In practice they are not always suitable to detect low levels of transcripts in situ. The technology of non-isotopic probes is described in the alternative protocol. The high sensitivity of autoradiography explains why radioactively labelled probes, are still widely used for ISH. For each particular application a radiolabel has to be selected as a function of its maximum energy of emission and resolution. 32p is highly energetic (1.71 Mev) and

leads to poor resolution. In contrast, the low energy of 3H (0.018 Mev) permits high resolution (subcellular localization) but imposes a very long exposure time (weeks). 35S (0.167 Mev) is a tradeoff between sensitivity and resolution, it gives good resolution (cellular level) with a convenient exposure time (5-10 days).

4.1.3. Length of probe As previously emphasised the access of the probe to the target is a function of the probe size and the fixation conditions. The optimum size for probes is within the range of 100-250 nucleotides.

4.2. Probe labelling The labelling methods described below, which all depend on enzymatic incorporation of modified nucleotides, are equally suitable with slight modifications for the addition of radioactively labelled or non-radioactively (see alternate protocol) labelled nucleotides.

4.2.1. Riboprobe preparation Transcription of single-stranded cRNA probes: methodology from Melton et al. (1984). Mix at room temperature in the order shown. (1) 4.0 /~1, 5 x transcription buffer (200 mM Tris-HCl pH 7.5 at 37°C, 30 mM MgCI2, 10 mM spermidine, 50 mM NaCI). (2) 2.0/.d, 100 mM DTT. (3) 0.8 tzl ribonuclease inhibitor: RNasin Promega, 25 U / ~ I , final concentration: 1 U//zl. (4) 4.0 pA, 2.5 mM each of ATP, GTP, CTP. Mix together equal amounts of 10 mM ATP, GTP, CTP stock solutions (provided by Promega) and H20. (5) 2.4 /~1, 100 /xM UTP (final concentration: 12 tzM). (6) 0.5-1 Ixg linearized plasmid template DNA. (7) 150 /~Ci [35S]UTP (Amersham, 1000 Ci/mmol). (8) 5-10 units (0.5-1 /xl), either SP6 or T7 RNA polyrnerase Promega. (9) Add DEPC-treated water to a final volume of 20 /~1. Incubate for 60 min at 37°C (T7 RNA polymerase), at 40°C (SP6 RNA polymerase).

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(10) After 30 min incubation, add 5-10 units more of either SP6 or T7. Removal of DNA template: following the RNA synthesis, add RQ1 DNase-RNase-free (Promega 1 U//zl) to a final concentration of 1 U / ~ g DNA. Incubate at 37°C for 15 min. (The completeness of the digestion of the template can be checked by running a small sample on 1% agarose gel.)

Probe extraction. (1) To transcription mix add (a) DT1~ (100 mM): 5.0 ~l; (b) t-RNA (10 mg/ml): 4.0 /xl; (c) H 2 0 DEPC treated: 170 ~1; (d) 3 M CHaCOENa pH 6.0:20.0/zl. (2) Vortex briefly, then add 100/zl of phenol. Vortex briefly, then add 100 /zl of chloroform. Vortex (30 s), spin (3 min) and remove bottom organic phase (discard). (3) Add 200/zl of chloroform to the aqueous phase, vortex, spin and remove organic phase. (4) Add 500 /xl of 95% ethanol (4°C) to the aqueous phase. (5) Spin briefly, place at -20°C for 2 h, then spin at 12 000 × g, for 20 rain (4°C). (6) Dissolve pellet in DEPC-treated water.

Alkaline hydrolysis size reduction of cRNA. Shorter cRNA probes (100-200 bp) give a higher ISH signal without any significant decrease in stability. Cox et al. (1984) described the following procedure for size reduction of cRNA by alkaline hydrolysis. However, if the cDNA insert is short (200-400 bp), it is unnecessary to carry out this procedure (go to step 9). (a) Dissolve pellet in 50 /zl water DEPC treated. (b) Add 50/.d of 0.2 M carbonate buffer pH 10.2 (80 mM NaHCO3, 20 mM NaECO3), made fresh or stored frozen. (c) Incubate at 60°C for time t in minutes: t = Li - Lf/0.11LiLf (Li = initial length in kb; Lf = final desired length in kb) (the rate K is approximately 0.11 strand scissions/kb/min). (d) Stop the reaction by adding 3/~l 3 M sodium acetate pH 6.0 and 5/~l 10% (v/v) glacial acetic acid. Ethanol precipitation. Add to the final 108 /xl mixture: (a) 92/~I H 2 0 DEPC treated; 20/xl 3 M CHaCO2Na pH 6.0. Mix, add 500 /xl 95% ethanol and precipitate

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at -20°C for 2 h or overnight. Spin at 4°C, 12000 × g for 30 min. Dissolve pellet in 20 /xl H 2 0 DEPC treated containing 20 mM DTF and 1 /xl RNasin. Reserve 0.1 /xl for counting and 1 ~zl to determine the size of hydrolysed RNAs (optional) in denaturing formaldehyde agarose gel. Store probe at -70°C until use.

4.2.2. cDNA probes labelling by nick-translation The nick-translation reaction is a rapid and easy method for producing uniformly radioactive DNA of high specific activity. Nick-translation uses a mixture of DNase and DNA polymerase I with labelled deoxynucleotides, to generate nicks into the DNA strands which are filled in by the polymerase. The reaction produces DNA fragments having a continuous range of sizes with a high specific activity. The DNA once labelled is usually purified from enzymes and salts before being used as a probe. Indifferently circular DNA, linearized plasmid or isolated insert, labelled by nick-translation can all be used for ISH. Generally the entire plasmid provides the best sensitivity, but in some situations the isolated insert will assume a better specificity.

4.2.2.1. Preparation. Prepare a DNAse I (BRL) stock solution 1 n g / m l in nick buffer-50% glycerol. Store at -20°C (stable for at least one year).

4.2.2.2. Nick translation reaction (for [35S]dCTP). To a microcentrifuge tube, add the following in sequence. (1) 5 /zl 10 × nick-translation buffer (0.5 M Tris-HC1 pH 7.2, 0.1 M MgSO4, 1 mM DTT, 500 # g / m l BSA). (2) 5 /xl of a mix of 0.5 mM dATP, dGTP, dTTP, prepared from 100 mM stock solutions of dATP, dGTP, dTTP (provided as lithium salt solutions, 100 mmol/l by Boehringer Mannheim). (3) 10/xl [3SS]dCTP (250/xCi) (1000 Ci/mmol, Amersham). (4) 1 /zl DNA polymerase I BRL (5 units). (5) 3/.d DNase I (3 ng).

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(6) 1 tzg cDNA. (7) H 2 0 is added to give a final volume of 50 /zl. Mix reagents and incubate for 3 h at 15°C. Stop reaction with 2 /zl 0.1 M ETDA pH 8.0. 4.2.2.3. Purification on N A C S P R E P A C column (BRL). (1) Hydration of column. (a) Clean column 3 times with 1 ml of 2.0 M NaCI in TE (pass through with a pipette) (TE is: 10 mM Tris-HC1 pH 7.2-1 mM EDTA). (b) Wash once with 1 ml of 2 M NaCI in TE (allow to drain by gravity). (c) Equilibrate 3 times with 1 ml of 0.2 NaCI in TE (pass through). (d) Then once again with 1 ml of 0.2 M NaCI in TE (allow to drain by gravity). (2) Load sample. Add sample (50/zl) to 450/zl of 0.2 NaCI in TE and apply to column (allow to drain by gravity). Collect effluent in microcentifuge tube No. 1. (3) Washing of column. Rinse microfuge tube containing reaction mix with 0.5 ml of 0.2 M NaCI and load this wash solution onto column (allow to drain by gravity), then wash column 3 times with 0.5 ml of 0.2 M NaCI. Effluents are collected in tubes No. 2, 3, 4 and 5. (4) Elute column. (a) With 250 ~1 of 2 M NaCI in TE, collect in tube No. 6. (b) Elute remaining probe with another 250 ~1 of 2 M NaCI in tube No. 7. Reserve Eppendorf No. 6 which contains the labelled probe, and count directly 0.1 /zl by scintillation counting. Add 20 mM DTT and store labelled probe at - 7 0 ° C until use. The range of DNA fragment sizes can be checked by running a sample in a 1.2% agarose gel. 4.2.3. Oligoprobe labelling by tailing Terminal deoxynucleotidyl transferase (TdT) catalyses the polymerisation of deoxynucleotides triphosphates onto the 3' end of single-stranded DNA. This labelling method generates oligoprobes of x,ery high specific activity. The added tail size depends on the amount of TdT used and the relative molar concentration of 3' ends versus labelled nucleotide. A longer tail is added using a lower amount of 3' ends and a large excess of labelled dNTPs.

Heat oligoprobe for 3 min at 65-70°C, chill on ice 2 min. Mix the following reagents in a microfuge tube. (1) 4/zl tailing buffer (5 × buffer, supplied by BRL with TdT is: 0.5 M potassium cacodylate (pH 7.2), 10 mM cobalt chloride, 10 mM DTT). (2) 4 /zl [35S]dATP: 40 /xCi (1300 Ci/mmol, NEN). (3) 50 ng oligonucleotide. (4) 40 units TdT BRL (10-20 units//zl). (5) Sterile DEPC-treated water is added to give a total volume of 20/.d. Mix, centrifuge briefly and incubate at 37°C for 1-2 h. Stop reaction by adding 1/zl EDTA 0.1 M pH 8.0. The protocol proposed here is established for the tailing of a 30 mer oligonucleotide. It can be optimized for oligonucleotides of a different length. As a rule, the ratio between pmol of [3sS]dATP and pmol 3' ends of oligonucleotide must be in the range of 4-6. Purify the probe from unincorporated nucleotides on NAP columns (Pharmacia) which are disposable columns prepacked with Sephadex G25. A complete step-by-step protocol, describing the fixed volumes needed for equilibration, washing and sample elution, is supplied with the columns. Oligoprobes purified by ethanol precipitation are also suitable for ISH. Complete removal of unincorporated nucleotides is not achieved by this method, but as a rule this does not significantly influence the background. This procedure operates well for oligonucleotides more than 20 nucleotides in length, but is poorly efficient for shorter probes. To the 20 tzl reaction mix, add 1 /zl of a glycogen suspension (10 mg/ml; special grade from Boehringer Mannheim) which is an effective coprecipitant without interfering with subsequent reactions, 30/zl H 2 0 DEPC treated, 5/zl 8 M LiC1 and 150/xl 90% ethanol. Mix, store at -20°C overnight and then centrifuge at 12 000 × g , 20 rain, 4°C. Wash the pellet with 80% ethanol and drain by inverting the tube. Dissolve the pellet in H 2 0 DEPC treated-20 mM DTI" and store at -70°C. Count 1 /zl of the eluted probe and calculate the specific activity.

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5. Hybridization The principles of hybridization in cell preparations or tissue sections are similar to those described for filter hybridization (Britten and Davidson, 1985). Hybridization is a reversible process and the stability of the duplexes formed is a function of the following: the temperature, the ionic strength, the number of base mismatches, the length of the probes and the concentration in formamide of solutions. Complementary strands of nucleic acids reassociate with a maximun rate at about 25°C below the melting temperature. Tm is the temperature at which half of the hybrids have dissociated into single strands. The rate of reannealing drops as the temperature approaches the Tm. The concentration of salt affects the rate of reassociation. Increasing ionic strength has a stabilizing effect on nucleic acid duplexes because of neutralization of the electrostatic repulsive forces between the negatively charged phosphate groups on opposing strands. Base composition also affects hybrid stability because GC base pairs are stabilized by three hydrogen bonds, whereas AT base pairs are stabilized by two; so duplexes with a high GC content are more stable. Furthermore poorly matched hybrids are less stable than fully base-paired duplexes and short hybrids than longer ones. Another important parameter affecting the stability of hybrids is the concentration of formamide in the solutions. This solvent destabilizes the double-stranded helix by disrupting hydrogen bonds. This permits to work at lower temperature and therefore providing a better morphology. Formamide decreases the Tm (each 1% in formamide concentration reduces the Tm by 0.35°C for RNA-RNA hybrids). From this it follows that the conditions of hybridization and washing stringency determine the degree of mismatching allowed between probe and target. Stringency is a concept referring to the degree to which reaction conditions favour the dissociation of nucleic acid duplexes. Stringency is increased by decreasing cation (sodium ion) concentration, increasing formamide concentration and increasing temperature. Usually hybridization is performed using low stringency conditions to favour hybrid forma-

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tion, and washing under increasing stringency to disrupt poorly matched probe-mRNA duplexes. Moreover the fact that RNA-RNA hybrids are more stable than RNA-DNA hybrids must be considered. The hybridization protocol proposed here has worked well in our laboratory with a wide range of DNA and RNA probes, but it must be underlined that there are no standard conditions of hybridization and washing stringency; each probe and tissue will require some individual assessment of the parameters to optimize the results. Slides processed immediately after fixation are washed in 1 × PBS twice (5 min each time). Slides stocked in 70% ethanol are rehydrated by dipping in 50% ethanol for 5 min, then in 1 × PBS: 2 changes of 5 min each. 5.1. Permeabilization step Designed to increase access of the probe to target sequences, the permeabilization step may be regarded as optional when small oligonucleotides are used. It differs for cells and cryosections which have been briefly fixed, and for paraffinembedded tissues which are more extensively fixed. 5.1.1. Using cells or cryosections (1) Incubate slides in 5 mM MgC12 in 1 × PBS: 10 min. (2) Then in 0.1 M glycine in 0.2 M Tris-HCl pH 7.4:20 min. (3) Wash briefly in DEPC-treated water, then postfix. 5.1.2. Paraffin sections Paraffin sections prepared from tissue fixed in PFA generally need a harsher treatment: this is achieved by a digestion with pronase or proteinase K. Different extents of digestion may be required for different tissues or fixative conditions. It is useful to try several concentrations in order to determine empirically the best compromise between tissue morphology and hybridization signal (overdigestion increases background). Pronase digestion: apply pronase (Calbiochem) 25-125/~g/ml in 0.1 M Tris-5 mM EDTA pH 7.6 for 10 min at room temperature. Pronase stock solution is prepared in 40 m g / m l in water, predi-

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gested at 37°C for 4 h to remove nucleases and stored in aliquots at - 2 0 ° C . Alternatively proteinase K digestion can be used: apply proteinase K (Boehringer Mannheim) 1 m g / m l in 0.1 M Tris-HCl 50 mM E D T A pH 8.0, at 37°C for 30 min. The buffer is prewarmed and the enzyme added just before it is used. A 1 m g / m l stock made in the same buffer is stored frozen in small aliquots. Proteinase K stock solutions do not need to be predigested. Digestion is stopped by rinsing slides in 0.1 M glycine in 0.2 M Tris p H 7.4; the incubation is continued for a further 20 min which should improve cell permeabilization. Briefly wash samples in DEPC-treated water (1 min). 5.2. Post-fix samples Post-fix 5 min in 4% PFA to prevent target diffusion. Then wash for 5 min in 1 x PBS. 5.3. Acetylation Acetylation of amino groups reduces nonspecific electrostatic binding of probes. Acetylate by immersing slides in 0.1 M triethanolamine-HC1 p H 8.0 (mix 591 ml H 2 0 , DEPC-treated, and 9 ml triethanolamine, adjust to p H 8.0 with HC1, use same day), stir vigorously for 2 min (magnetic stirrer) and then add undiluted acetic anhydride to 0.25% (v/v). Stir for 10 min. Then wash slides in 2 x SSC for 5 min. 5.4. Prehybridization (optional) Rare probes possess sequences associated with relatively high backgrounds. For these particular probes a prehybridization step can improve the signal. The components of the mix used are intended to saturate sites in the samples that bind nucleic acid non-specifically. To 7 volumes of the hybridization stock buffer (see below) add 2 volumes of DEPC-treated water, mix thoroughly, spin and apply 20 /zl of the mix to the samples. Cut a small square from Parafilm and place inner surface on a drop of hybridization buffer. Incu-

bate for 1 h at 45°C in a moist chamber. Then carefully remove the Parafilm with stainless steel tweezers, blot off buffer against Whatman paper and proceed to the hybridization step. 5.5. Hybridization Prepare hybridization stock Harper and Marselle, 1987):

buffer

(from

Final concentration in the diluted buffer: 9 ml 50% 1.440 ml 2×SSC 0.390 ml 0.1M 0.900 ml 1 mg/ml

Deionized formamide 25 × SSC DTT (4.8 M) Yeast tRNA (20 mg/ml RNase-free) Sonicated salmon sperm 1.800 ml 1 mg/ml DNA (10 mg/ml) BSA (80 mg/ml RNase-free) 0.450ml 2 mg/ml

Store 150/zl aliquots (for 10 slides) at -20°C. Add 2 volumes of probe (diluted in DEPCtreated water at a concentration of 1-2.2 × 10 6 c p m / 1 0 / z l ) to 7 volumes of hybridization buffer to achieve the required final hybridization mix (see scheme above) and probe concentrations (0.2-0.5 x 106 c p m / 1 0 / z l ) . Denature diluted cDNA probes in water bath at 95°C for 10 min and oligoprobes at 70°C for 5 min and then chill on ice for 5 min. Heat riboprobes at 90°C for 3 min and maintain at 50°C. Apply 5 /zl of hybridization mix per cm 2 of coverslips used. Slowly lay the coverslips over the drop of mixture to minimize bubbles. Do not press down. Arrange slides on a rack and place inside a microwave oven plastic box humidified with a 50% formamide-2 x SSC solution. Tightly close the box and seal it with Saran Wrap. In a precisely regulated convection oven incubate overnight at 37-45°C for c D N A and oligoprobes or at 45-55°C for riboprobes. 5.6. Post-hybridization washing The goal of post-hybridization washes is to obtain a signal of high specificity by removing

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mismatched hybrids and non-specifically bound probe. The stringency required for an optimal signal-to-noise ratio is determined by trial and error, adapting temperature, salt and formamide concentrations of the washing solution. The acceptable level of background is also a function of the intensity of the signal and the length of the intended exposure (a long time exposure necessary to detect a weak signal will require extensive washes to get a very low background). Remove the coverslips by immersing slides in 50% formamide-2 x SSC at 37°C in Coplin jars. Coverslips will detach in a few seconds. If necessary agitate the slides gently up and down (do not manually remove them). Slides must not dry at any stage during the washing steps (it will result in a high background) and washes must be carried out under gentle agitation in a large volume of wash solution.

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(2) 50% formamide-1 x SSC-10 mM DTI" at 37°C, 30 min; (3) 1 x SSC: three changes of 10 min each at room temperature; (4) sometimes it may be necessary to add a further step: 0.1 x SSC, 3 times (10 min each), at room temperature or 37°C; (5) air dry the slides and detect hybridized probe by autoradiography.

5.6.3. Oligoprobes Wash slides successively in: (1) 1 x SSC: two changes of 30 min each at room temperature; (2) 1 x SSC: two changes of 30 min each at 40°C; (3) 0.1 x SSC at 37-45°C, twice, 30 rain each time.

5.6.1. RNA probes Wash with gentle agitation in: (1) 50% formamide-2 x SSC-10 mM DT-I" at 50°C: 3 changes, 30 min each. (2) Blot around sections, then remove unhybridized single-stranded probes using 200 /xl/sample of a RNase A / R N a s e T1 digestion solution consisting of 100 /zg/ml RNase A and 500 units/ml RNase T1 in 2 x SSC. Place slides in a moist chamber and incubate at 37°C for 30 min. RNase stock solutions are: (a) RNase T1 (BRL, 900-3000 units//zl); (b) RNase A (BRL), prepare a 5 m g /ml stock solution in 2 X SSC and deactivate DNases by heating the solution for 5 min at 100°C. (3) Rinse slides briefly in 2 X SSC. (4) Wash again in 50% formamide-2 × SSC-10 mM DTT at 50°C twice, 30 min each time. (5) Wash in 0.5 x SSC at room temperature: two changes of 15 min each. (6) Remove excess SSC and air dry slides until ready for dipping in emulsion.

5.6.2. DNA probes Wash using: (1) 50% formamide-2 x SSC-10 mM DTI" at 37°C three changes 30 min each;

6. Detection of hybridized radioactive probes

6.1. Film autoradiography Film autoradiography gives some insight into the tissue background, distribution and strength of the hybridization signal. For this purpose, tape slides onto a piece of cardboard. Expose slides to Hyperfilm /3max and develop after 2-4 days of exposure (for 35S-labelled probe).

6.2. Autoradiography Slides must be processed in the dark or more conveniently under safelight condition using filters adapted for autoradiography.

6.2.1. Preparation of emulsion Heat Kodak NTB2 emulsion at 42°C in a water bath for 30 min. When melted, dilute with an equal volume of 42°C sterile water. Mix gently and avoid creating bubbles. Aliquot in 20 ml glass or plastic vials. Store in light-tight boxes at 4°C (up to 3 months). Alternatively Amersham LM1 undiluted or Ilford K5 diluted 1:1 (v/v) emulsions, can be used.

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6.2.3. Development of slides at 15°C under safe-light conditions The different solutions and the slides must be equilibrated at 15°C to prevent wrinkling of the emulsion. This can be conveniently achieved by removing about 1.5 h before developing the developer, fixer and rinsing water from the refrigerator, where they are stored at 4°C. Slides are taken out only 30 min before developing. Develop as follows: immerse slides in Kodak D19 developer for 2.5 min, transfer into water for 30 s, then into Kodak Unifix for 5 min. Rinse slides for 15-20 min under gently running cool tap water.

6•2•2• Handling At the time of autoradiography, melt an aliquot of emulsion in a water bath at 42°C, for 30 min. Pour melted emulsion into a dipping chamber (Dip miser from Electron Microscopic Sciences). Slowly dip the slides (for 2 s) and withdraw them from the emulsion, wipe the back and allow them to dry vertically on a rack slide holder for 20 min. Carefully, handling slides by their edges, place them in slide incubation chambers (Scienceware). These are black light-tight boxes with a compartment at the top containing silica gel desiccant. Wrap boxes with two opaque film bags, and store overnight at room temperature, before cooling to 4°C. Exposure time depends on the radioactive label used, the level of mRNA per cell, and the amount of probe used; therefore, optimal exposure times must be empirically determined. Therefore hybridization experiments are usually set up in duplicate or triplicate. For samples hybridized with 35S-labelled probes time exposure is in the range of 5-15 days.

6.3• Staining of slides Many nuclear a n d / o r cytoplasmic dyes can be used (haematoxylin, eosin, Giemsa, methylene blue). Staining procedures that use long incubations at low pH or relatively high concentration of oxidant should be avoided, because this bleaches autoradiographic grains. Staining procedures that

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Fig. 1. PDGF B-chain gene expression by alveolar lung macrophages in idiopathic pulmonary fibrosis lung biopsy. ISH was performed using a 35S-labelled antisense PDGF B-chain riboprobe. Magnification x 1160.

J.M. Vignaud et al. /Journal of lmmunological Methods 174 (1994) 281-296

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Fig. 2. Normal human blood monocytes,purified by Hypaque-Ficollcentrifugation and adherence to a plastic dish, were activated (lipopolysaccharide 10/zg//.d) in culture (24 h in DMEM) before being evaluated for PDGF A-chain gene expressionby ISH using a 35S-labelled antisense PDGF A-chain riboprobe. Dark-field examination. Magnification: x 1160.

produce too much contrast may interfere with visualization of grains by bright- and dark-field microscopy. 6. 4. Microscopy

Examination with dark-field optics gives the general pattern of labelling: developed silver grains appear as white dots on a black background. Switching over to bright field permits the determination of labelled cell type, by displaying a cytoplasmic deposition of silver grains. Negative controls should be screened thoroughly to prevent misinterpretation of labelling (Figs. 1 and 2).

7. Controls

Controls are in all cases an integral part of the technique. Unexpected homologies between short regions within a probe and unknown target sequences, non-specific binding linked to other properties of the probe or autoradiography arti-

facts may produce convincing false-positive results. A well-known artifact is the binding of electronegative probes to highly charged cationic proteins such as those of eosinophilic granules. When using R N A probes the sense transcripts (non-complementary) provide the best control because they have the same activity, concentration and fragment length as the antisense probe (complementary). When working with a c D N A probe it is necessary to select a heterologous probe with the same characteristics and compare the expression patterns. Prehybridization treatment with RNase is not totally reliable, because it is difficult to remove cellular m R N A completely from sections and the probe can be degraded by residual RNases. Hybridization with a different fragment of the specific sequence and prehybridization with the unlabelled probe may also be used. Additionally, comparison with external data (Northern blot and immunohistochemical studies) provides useful information. Furthermore for the initial experiments it can be helpful to process cells or tissues that express the gene of interest at high levels in the Northern blot.

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8. Alternative protocol: non-radioactive in situ hybridization procedures The limitations associated with the use of radioisotopes (personal safety, disposal problems, reduced stability, long time exposure and cost) have stimulated the development of non-isotopic probes. Although their sensitivity does not fully match those of radioactive probes, they are safe, give good resolution and allow results to be obtained in a short time. Numerous non-enzymatic and enzymatic procedures have been described for labelling the probes. Non-enzymatic methods include chemical modification and cross-linking of proteins to DNA. These procedures, generally inexpensive and simple to perform are, however, less sensitive for ISH applications than enzymatic methods. In these procedures biotin-, digoxigenin- or fluorescein-labelled nucleotides are incorporated into DNA and RNA probes. These haptens are attached with an atom spacer arm to a nucleotide (most often dUTP or UTP). These modified nucleotides serve as substrates for enzymes (e.g. DNA polymerase, RNA polymerase) and are incorporated into DNA or RNA probes by substituting them for thymidine or uracil. The suitability of hapten-labelled nucleic acid molecules as hybridization probes depends on two parameters: the degree of label incorporated and the length of labelled fragments. Hybrids of hapten-labelled probes have a lower Tm than their radioactive counterparts. The more hapten residues incorporated, the greater the decrease in the Tm. Therefore the optimal sensitivity is a compromise between detection sensitivity and hybridization efficiency. In general, probes with 2532% substitution give the highest sensitivity. To achieve this, the degree of substitution can be fine tuned by adding a small amount of the corresponding unlabelled nucleotide (e.g. dTTP for digoxigenin-dUTP) in the labelling reaction (Chan et al., 1985). This is most important when digoxigenin and fluorescein labelling is utilized. The sensitivity of the method is also to a large extent a function of the length of the spacer arm: the shorter the linker, the more efficient is the incorporation of the labelled nucleotide into the probe, but also the less efficient is the recognition

by the detection system. For ISH applications we have found that 11- to 16-atom spacer arms perform best. However, oligoprobes labelled by tailing are exempt from these considerations. They achieve their sensitivity, in our hands, using modified nucleotides with a 21-atom spacer arm. The hybridized biotinylated probes are most often detected by the binding of streptavidin-alkaline phosphatase conjugates. Fluorescein- and digoxigenin-labelled probes are visualized using indirect labelled antibody methods. Furthermore, hapten-labelled hybrids may also be detected with an electron microscope using secondary antibodies conjugated to colloidal gold. Digoxigenin-, fluorescein-, and biotin-labelled probes are equally suitable for ISH applications. We have not observed a significant difference between them in terms of sensitivity and specificity. Theoretically digoxigenin and fluorescein circumvent the high backgrounds caused by endogeneous biotin that is present in some tissues (e.g. liver). As the antibody or streptavidin solutions used to visualize labelled hybrids are not consistently RNase-free, they should be DEPC treated. Apart from probe labelling, the ISH protocol using non-radioactive probes differs from the previous procedure, in that the detection system is immunohistochemical and the washes for some probes must be less stringent. Furthermore, in addition to the panel of controls previously proposed for isotopic ISH, if an immunoenzymatic method is used for detecting the hapten-labelled hybrid, samples checked for residual endogeneous enzymatic activity must be added. Biotin-modified nucleotides may be purchased from different companies (BRL, Enzo Biochem, Sigma, Clontech) sold either as part of a labelling kit or separately. Digoxigenin- and fluoresceinlabelled nucleotides are supplied by BoehringerMannheim. 8.1. Non-radioactive labelling procedures

The labelling procedures described here are set up for bio-ll-dUTP and UTP; they also work well using either digoxigenin-ll-dUTP and UTP or fluorescein-12-dUTP and UTP. Step-by-step detailed protocols, fully optimized for digoxi-

ZM. Vignaudet al./Journal of lmmunological Methods 174 (1994)281-296 genin- and fluorescein-modified nucleotides, are available from Boehringer-Mannheim. 8.1.1. Synthesis of biotinylated RNA probes Bio-ll-UTP is incorporated into RNA with a weak efficiency, so the yield of synthetised labelled RNA is lower than that obtained with the radioactive labelling transcription reaction. 8.1.1.1. Reaction components. Add the following reaction components at room temperature, in the order shown: (a) 5.0/zl of 5 x transcription buffer (this 5 x buffer is: 200 mM Tris-HCl pH 7.5-30 mM MgCI2-10 mM spermidine); (b) 10.0 tzl of 2.5 mM ATP, CTP, GTP in 20 mM Tris-HCl pH 7.5 (prepare from 10 mM stock solution Promega); (c) 1.0 /zl of 0.5 M DTI'; (d) 10.0 Izl 5 mM bio-ll-UTP (0.5 mg Bio-ll-UTP Sigma dissolved in 113 #1 of 50 mM Tris-HC1 pH 7.5 gives a 5 mM stock solution); (e) 1-2 /zg linearized plasmid; (D 1 txl RNasin (25 units/tzl stock); (g) 30 units of either T7 RNA polymerase or SP6 RNA polymerase (3 /zl); (h) Increase volume to 50 lzl with DEPC-treated water. Incubate at 40°C for 1.5 h. After 30 min incubation add 2 /zl more of either SP6 or T7 polymerase. 8.1.1.2. DNA template. Then remove DNA template as described in 4.2.1. 8.1.1.3. Purification. Purify the probe from unincorporated nucleotides by gel chromatography on the 'Quick Spin G-50 Column for Biotinylated RNA' supplied by Boehringer-Mannheim, or by ethanol precipitation. A labelled RNA probe is stable for one year when stored at -70°C provided that all solutions used are RNase-free. 8.1.2. Labelling of DNA with bio-ll-dUTP by nick-translation To a microcentrifuge tube, add in the following sequence: (a) 5 #1 10 X nick-translation buffer (0.5 M Tris-HC1 pH 7.2, 0.1 M MgSO4, 1 mM DT/', 500/zg/ml BSA); (b) 5 /zl of a mix of 0.5 mM dATP, dCTP, dGTP (from stock solutions of 100 mmol/1 supplied by Boehringer Mannheim); (c) 4 /xl bio-ll-dUTP; (d) 1.2 Izl DNA poly-

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merase I, BRL (5 units); (e) 3/~1 DNase I, BRL (3 ng, from stock solution 1 ng//xl); (f) 1 /zg of the eDNA to be labelled; (g) H 2 0 DEPC-treated to 50 ~1. Mix reagents and incubate reaction for 2 h at 15°C. Stop reaction with 2 /zl 0.1 M EDTA pH 8.0. Purification of biotinylated DNA probe: phenol extraction of biotinylated DNA should always be avoided due to the possible partitioning of the probe into the phenol layer. The labelled probe may be separated from unincorporated nucleotides by ethanol precipitation or spin column methods. The biotin-labelled probe is stable for at least one year when stored in TE buffer or in hybridization solution at -20°C. 8.1.3. Tailing of oligonucleotides probes with bio21-dUTP Heat oligoprobe for 3 min at 65-70°C; chill on ice for 2 min. Mix the following reagents in a microfuge tube: (1) 4 lzl tailing buffer (5 x buffer, supplied by BRL with TdT is: 0.5 M potassium cacodylate (pH 7.2), 10 mM COC12, 10 mM DTT); (2) 4 /1.1 bio-21-dUTP stock solution 0.5 mM (Calbiochem); (3) 50 ng oligonucleotide; (4) 40 units TdT BRL(10-20 units/p.1); (5) Sterile DEPC-treated water to give a total volume of 20/zl; (6) Optional. For further purification of the labelled probe on Sepharose G 25 column add: 1 /zl of [3H]dATP (10-25 Ci/mmol, NEN) diluted 1 : 5 (v/v) as tracer. Mix, centrifuge briefly and incubate at 37°C for 1.5 h. Stop reaction by adding 1/zl EDTA 0.1 M pH 8.0. Add 5/zg yeast tRNA. Purify the probe from modified unincorporated nucleotide either on NAP columns (as described in 4.2.3) or by ethanol precipitation (4.2.3). Labelled probes are stable for at least one year at - 2 0 ° C . Note that this method adds a very short tail of an average length of only two haptens since the biotin-labelled dUTPs are added inefficiently to the oligonucleotides.

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8.2. Hybridization Proceed in the same manner as described for hybridization with radioactive probes in Sections 5.1.-5.6. For 20/~1 of final hybridization mix use either 8-10 ng of riboprobe, or 20-30 ng of labelled cDNA probe or 1-5 ng of biotinoligoprobe. Some modified probes may require less stringent conditions than their radioactive counterparts and this can be achieved, initially, by decreasing the temperature of the post-hybridization washes.

8.3. Colorimetric detection of hybridized biotinlabelled probes Standard antibody procedures (e.g., indirect) or the streptavidin-biotin method can be employed for the hapten detection. For details of the immunohistochemical procedures see our companion paper in this issue: Immunohistochemical methods for studying mononuclear phagocytes in tissue sections. It should be noted that streptavidin and antibody solutions are not free of RNases. They must thus be treated with 0.04% DEPC. To avoid denaturation of antibodies, DEPC should not be destroyed by heating before the use of the solutions. It is our opinion that the streptavidin-alkaline phosphataseBCIP/NBT method gives the highest sensitivity. Changing any component of this combination invariably results in decreased sensitivity. The solutions containing BCIP and NBT that are precipitated by DEPC cannot be RNase inactivated.

8.3.1. Reagents Blocking solution: 3% gelatine, 3% BSA, 0.3 M NaCI, 0.1 M Tris-HC1 pH 7.5, 0.005% sodium heparinate. Add 0.04% DEPC just before use. Nota bene: Do not use nonfat dried milk as a blocking reagent: this solution is very rich in RNases. Buffer A: 0.1 M Tris-HCl pH 7.5, 0.3 M NaC1, 3% BSA (RNase free). Add 0.04% DEPC just before use. Buffer B: 0.1 M Tris-HCl pH 9.5, 0.15 M NaC1, 50 mM MgC12 with 0.06% levamisole for blocking endogeneous alkaline phosphatase.

BCIP/NBT stock solutions should be stored in the dark at -20°C and used within 3 weeks. (a) BCIP stock: 50 mg/ml in N,N'-dimethylformamide (Merck) prepared from BCIP disodium salt (Sigma). (b) NBT stock: 75 mg/ml NBT in 70% N,N'-dimethylformamide. Dissolve NBT (Sigma) in concentrated dimethylformamide, then bring to 70% with sterile water.

8.3.2. Procedure After post-hybridization washes at the desired stringency maintain slides in 2 x SSC until colorimetric detection. Wash slides in buffer A, for 5 min. Incubate in blocking solution for 1 h (300 /~l/sample) at room temperature. Blot excess solution onto laboratory wipe. Apply alkaline phosphatase conjugates (BRL) dissolved in a 1/500 dilution (0.2 ~ g / m l ) of buffer A for 2 h, at room temperature. Pour off the conjugate solution and wash slides 3 times (5 min each), using gentle agitation with wash buffer B. Develop colour by incubating slides in the dark with NBT/BCIP substrate solution (500/xl/sampie), in a humidified light-tight box, checking from time to time for colour development. Substrate solution is prepared just before use by mixing: 7.5 ml buffer B, 33/~1 NBT stock solution and 25/zl BCIP stock. Development of colour in the target mRNA generally requires several hours (3-6 h). If the background is light after this time and more development is needed, incubation can be continued overnight. Stop reaction in gently running tap water for 20 min. Mount in a water-soluble moutant. Allow to dry and examine microscopically. It should be noted that the dense blue precipitate is soluble in non-aqueous mounting media.

9. Combined ISH and immunohistochemistry

Immunohistochemistry and ISH may be performed sequentially to detect mRNAs and proteins in the same cell, or in different cell populations present in the same tissue section. For this

J.M. Vignaud et al. /Journal of lmmunological Methods 174 (1994) 281-296 purpose m R N A s and antigens must be preserved throughout the technique and two different signals must be generated and retained. I S H should be p e r f o r m e d first followed by immunohistochemistry when the antigen is not denatured during ISH. However, if antigen is lost during the I S H procedure, it will be necessary to perform immunohistochemistry first, even if this is generally correlated with a weaker signal. Shivers et al. (1986) demonstrated that the addition of 0.04% D E P C in primary and secondary antisera protects m R N A s against enzymatic degradation during the immunocytochemical step. For further discussions see Brahic et al. (1984), Shivers et al. (1986).

10. Quantitative in situ hybridization The n u m b e r of silver grains developed after in situ hybridization is proportional to the n u m b e r of hybrids formed which, at saturation, is equivalent to the n u m b e r of m R N A sequences to be detected. This provides the basis for using the technique in a quantitative manner. For quantification at the cell level 3H- or t25I-labelled probes allowing single-cell resolution are used. The resuiting autoradiograms are analysed by measuring diffuse integrated optical densities in a computerassisted image analysis system. See Davenport and Nunez (1990) for a critical discussion of the method.

11. Buffers and reagents 11.1. Buffers All buffers are p r e p a r e d according to Sambrook et al. (1989), except for 10 × PBS which is: 0.07 M N a 2 H P O 4, 0.03 M N a H z P O 4 , 1.3 M NaCI, p H 7.4. 11.2. Reagents and equipment Chemicals are purchased from different companies: in each case use the highest grade of purity available. Equipment for autoradiography: (a) Super Du-

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plex Safelight, V W R TM72882-10 with F D Y Top and F D W bottom filters. (b) Incubation chambers for autoradiography: Scienceware 44 182. (c) Dipping chambers: Dip miser, Electron Microscopy Sciences 70 510. (d) NTB-2 emulsion: Eastman Kodak 165-4433. (e) Kodak D19: Eastman Kodak 502-7065. (f) Kodak Unifix: Eastman Kodak 5011036. 11.3. Working with toxic reagents Paraformaldehyde is a potential carcinogen and irritating to eyes and skin. It must p r e p a r e d in a fume hood. D E P C decomposes in ethanol and carbon dioxide when added to water. However, before decomposition it can react with ammonia and potentially with the body's endogeneous a m m o nia, to form urethane, a powerful carcinogen. Phenol can cause severe burns with toxic effects if inhaled or absorbed through the skin. Xylene and chloroform should be handled with care.

Acknowledgements This work was supported by a grant from the Philippe Foundation and l'Association pour la Recherche Contre le Cancer.

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situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485. Davenport, A.P. and Nunez, D.J. (1990) Quantification of radioactive mRNA in situ hybridization signals. In: In Situ Hybridization Principles and Practice. Oxford University Press, New York, p. 95. Hames, B.D. and Higgins, S.J. (1985) Nucleic Acid Hybridization: A Practical Approach. IRL Press, Oxford - Washington, DC. Harper, M.E. and Marselle, L.M. (1987) RNA detection and localization in cells and tissue sections by in situ hybridization of 35S labelled RNA probes. Methods Enzymol. 151, 539. Keller, G.H. and Manak, M.M. (1989) DNA Probes. Stockton Press, New York. Lawrence, J.B. and Singer, R.H. (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 13, 1777. McAllister, H.A. and Rock, D.L. (1985) Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization. J. Histochem. Cytochem. 33, 1026. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035.

Moench, T.R., Gendelman, H.E., Clements, J.E., Narayan, O. and Griffin, D.E. (1985) Efficiency of in situ hybridization as a function of probe size and fixation technique. J. Virol. Methods 11, 119. Perbal, B. (1988) A Practical Guide to Molecular Cloning. Wiley Interscience, John Wiley and Sons, New York. Polak, J.H. and McGee, J. (1990) In Situ Hybridization Principles and Practice. Oxford University Press, New York. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning - A Laboratory Manual. Coldspring Harbor Laboratory Press, Cold Spring Harbor, NY. Shivers, B.D., Harlan, R.E., Pfaff, D.W. and Schachter, B.S. (1986) Combination of immunocytochemistry and in situ hybridization in the same tissue section of rat pituitary. J. Histochem. Cytochem. 34, 39. Valentino, K.L., Eberwine, J.H. and Barchas, J.D. (1987) In Situ Hybridization: Applications to Neurobiology. Oxford University Press, Oxford. Vignaud, J.M., Allam, M., Martinet, N., Pech, M., Plenat, F. and Marfinet, Y. (1991) Presence of platelet-derived growth factor in normal and fibrotic lung is specifically associated with interstitial macrophages, while both interstitial macrophages and alveolar epithelial cells express the c-s/s proto-oncogene. Am. J. Respir. Cell. Mol. Biol. 5, 531.