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Fundamental principles of in situ hybridization.

J N Wilcox J Histochem Cytochem 1993 41: 1725 DOI: 10.1177/41.12.8245419 The online version of this article can be found at: http://jhc.sagepub.com/content/41/12/1725

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0022-1554/931$3.30

The J o u d of Histochemistry and Cytochemistry Copyright 0 1993 by The Histochemical h i c t y , Inc.

Vol. 41, No. 12, pp. 1725-1733, 1993 Printed in UXA.

Workshop

I
Fundamental Principles of In Situ Hybridization'
JOSIAH N. WILCOX~
Department of Medicine, Division of Hematology/Oncology, Emory University, AtZanta, Georgia. Received for publication September 17, 1993; accepted September 1, 1993 (3W3159).

In situ hybridizationprovides invaluableinformationregarding 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. This review provides a starting point for those who wish to begin using in situ hybridization in their own laboratories. The procedure outlined here is based on 35S-labeledriboprobes and has been used with many probes and tissues with a greater than 9O0/o success rate on the first hybridization. The importance of appropriate controls is stressed. Clusters of silver grains

after hybridizationdo not n e d y indicate specific mRNA

localization. Regions of the tissue rich in nudei often appear to cause spurious binding of probes and have high backgrounds often mistaken as positive signals. The most acult aspect of in situ hybridization is not to get dusters of silver grains on the slide but rather to do the appropriate controlled experiments to ensure that the signal is real and is not due to some artifactual binding of the probe to the tissue. ( J Hisrochem Cyrochem 41:1725-1733, 1993)
KEY WORDS:In

situ hybridization; mRNA; Riboprobes.

Introduction
In situ hybridization is an invaluable tool for the examination of gene expression. In situ hybridization is very similar to Northern blots and depends on the hybridization of a labeled nucleic acid A probe (RNA or DNA) to a complementary sequence of " . The two techniques differ in that the starting material for a Northern blot is a tissue digest, whereas the primary material for in situ hybridization is a histological tissue section. All of the cell relationships are lost with Northern blots and mRNA levels are averaged from all of the cells contained in the original sample. However, in situ hybridization is exquisitely sensitive and can detect the amount of mRNA contained in a single cell. Furthermore, since in situ hybridization is a histological technique, cell relationships are maintained and it is possible to precisely identify cell types expressing the gene of interest. Thus, potentially important interactions between cells that express different proteins may be uncovered. A major advantage of in situ hybridization is that it allows the maximal use of tissues that may be in short supply (i.e., clinical biopsies, embryos, cultured cells). A tissue digest from a surgical biopsy might yield sufficient RNA for one or two Northern blots. These Northern blots would only yield information regarding the presence or absence of mRNAs without any other information as to the cellular source of that RNA. Although Northern blots can be probed multiple times for different " s A, in fact this may be limited to four or five different probes at most, as the transfer

membranes break down and there can be some RNA loss with repeated stripping of the blots. However, literally hundreds of different hybridizations can be performed on the same piece of tissue with in situ hybridization. A single surgical biopsy of a human atheroscleroticplaque obtained at carotid endarterectomy, for example, might yield up to 1000 tissue sections, each of which can be used for a separate hybridization. Kept at - 70C with desiccant, these tissue sections can be stored for more than 6 years without significant loss of the hybridization signal (unpublished observations). It is therefore possible to make libraries of tissues, stored as sections in the freezer, that can be probed in the future as new probes become available or new questions arise. The repeated hybridization of these tissues with multiple probes over a long period of time also allows the eventual serial reconstitution of a tissue with all of the information concerningmRNA content, cell identity, and regional localization available for study. There are many different ways to do in situ hybridization including probing with cDNAs, cRNAs, or synthetic oligonucleotides. There are also multiple ways to label these probes using radioactive ( 3H, 32P, 33P, 3%) or non-radioactive (biotin, alkaline phosphatase, digoxigenin, fluorescence) nucleotides. Over the past 10 years we have compared most of these methods in my laboratory and have come to the conclusion that 35Sriboprobes represent the most sensitive method for the detection of mRNA in tissue sections. A comparison of different labeling strategies for in situ hybridization ranked according to their relative sensitivity is shown below. Riboprobes > cDNAs > Synthetic oligomers 35S> 32P> 3H >> Biotinldigoxigenin Frozen > Paraffin
1725

Supported by NIH Grant HL47838 and by a grant from the Emory University Department of Medicine Development Fund. Correspondence to: Box AJ, Hematology, Rm. 1115 WMB, 1639 Pierce Dr., Emory U,, Atlanta, GA 30322.

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1726

WILCOX

This review presents an overview of in situ hybridization, first introducing the various uses of the procedure in the analysis of local gene expression in tissues and then focusing on some of the more technical aspects of the procedure dealing with the choice of probe and the question of appropriatecontrols. It concludes with an overview of an in situ hybridization protocol used in my laboratory, which is based on 3%labeled riboprobes that we have used with great success and reliability on a variety of different tissues ranging from surgical biopsy specimens to animal tissues. To date, we have used this protocol with more than 1000 different tissues and more than 100 different probes, without modification. The success rate of this method with a new probe and tissue is greater than 90% on the first series of hybridizations, thus providing rapid results without the necessity of a series of experiments establishing optimal conditions individualized for a specific tissue or probe. It is hoped that this protocol may provide a starting point for those who wish to begin in situ hybridization in their own laboratories. The method we employ for in situ hybridization is basically a modification of the original protocol for radiolabeled riboprobes presented by Cox and colleagues (2), and has been used by our lab for many different projects (7,9,12,14,15,17-19). This method can be adapted for synthetic oligomers (40-50-mers), cultured cells, or paraffin-embedded tissues.

loss of tissue from the slides during hybridization. Excessive cross-linking of the tissue by paraformaldehydemay reduce potential binding to charged molecules in the slide coating which are responsible for tissue adhesion. Overfixation also reduces the in situ hybridization signal, presumably by limiting probe access to cellular As. This can sometimes be recovered by increasing the concentration or the time in proteinase K (1). Fresh frozen tissue without fixation can be used for in situ hybridization. Tissue should be rinsed in saline or PBS and frozen in liquid nitrogen in OCT blocks as outlined above. Although not optimal, tissue can also be snap-frozenwithout an embedding matrix and used for hybridization. The main determinant of the fixation method is based on the tissue morphology after hybridization. Some tissues, such as lung, are not suitable for frozen sections and must be prepared as paraffin blocks for the best cell morphology. Paraffin-embedded tissues can be used for in situ hybridization with an approximate 25% mRNA signal loss. This loss of signal intensity is acceptable, especially for tissues in which frozen sections are not practical. Additional details on using paraffin-embedded sections are covered below.

Sectioning o Frozen Tissue f

The tissues are sectioned on a cryostat to 5-10-pm thickness, thaw-mounted on coated slides (0.2 N NaOH-PLL, Vectabond, or Fisher SuperfrostlPlus), and immediately re-frozen by placing the slides in plastic slide boxes kept in cryostat or on dry ice. After completion of the sectioning the slides are stored at - 70C with Humi-Cap desiccant capsules (United Desiccants; Pennsauken, NJ) in the slide boxes. The mRNA on the slides is stable when stored in this fashion, and we have used tissue sections for up to 8 years after sectioning with excellent hybridization results. This can be very valuable for working with human tissues or small tissue biopsy specimens and enables the researcher to conduct multiple studies on related adjacent sections over the years as new probes become available. In addition, it is possible to store hundreds of sections typical of a particular animal model and to continue to examine the expression of additional mRNAs without having to prepare new tissues. Given that the major effort of performing in situ hybridization lies in the preparation of the tissue sections, it is relatively easy for a researcher to hybridize tissue sections from a wide range of animal models, human biopsy specimens,and normal tissues when probing with new cDNAs. This type of random screening does not take very much additional time but may yield a wealth of information and suggest new directions for research.

Tissue Preparation
1. Remove tissue and rinse in PBS or saline.
2 . Immerse in 4% paraformaldehyde-0.1 M sodium phosphate buffer, pH 7.4, at 4C for 1-3 hr. Try to avoid overnight fixation if possible, as this

3. 4.

5.

6.

causes problems with keeping the section on the slide during the hybridization procedure. Immerse in sterile 15% sucrose-PBS solution 3 hr-overnight at 4C. Embed tissue in OCT (Miles; Elkhart, IN), M1 (Lipshaw; Pittsburgh, PA), or any other convenient embedding matrix for frozen sectioning. Tissue should be oriented in the block appropriatelyfor sectioning.Note the tissue number on the block directly and indicate which face of the block should be sectioned. Freeze block with tissue in liquid nitrogen. Place the bottom third of the block in the liquid nitrogen, freeze until all but the center of the block is frozen, and continue freezing on dry ice. Store at -70C in a sealed container or wrapped in foil. Ship on dry ice if necessary.

SLide Preparation/Tissue Adberence

One of the biggest problems with in situ hybridization is keeping the tissue sectionson the slide during the hybridization and washing procedures. A variety of slide coatings have been tested (Tables 1 and 2). and although some seem to be better than others, none is absolutely perfect. We have found that the best coatings for tissue retention include poly-L-lysine and Vectabond (Vector Laboratories; Burlingame, CA). Vectabond slide coating is a proprietary modification of the amino-acyl silane procedure and has given excellent in situ hybridization results with good tissue morphology after the hybridization procedure. We have recently begun to use SuperfrostlPlus microscope slides (Fisher Scientific; Pittsburgh, PA) for all of our frozen tissue sectioning. All three methods of slide coating appear to have similar properties regarding tissue morphology/retention on the slide after the hybridization procedure (lible 2). The advantage of Superfrost/Plus slides is that they require no preparation time in the laboratory and are competitivein terms of cost when technician time and reagent expenses are considered. Sections placed on poly-L-lysine, Vectabond, or Superfrost/Plus slides do not require coverslips during hybridization. With gelatin-coated slides

Paraformaldehyde is prepared by dissolving the appropriate amount of powdered paraformaldehyde (Polysciences; Warrington, PA) in 0.1 M phosphate buffer and heating to 80C. The subsequent solution is filtered with Whatman #1 paper after cooling and is stored at 4C until use. The paraformaldehyde should be freshly prepared at least weekly to prevent the formation of degraded aldehyde byproducts which may cause an increase in the autoradiography background. The optimal period for fixation is 1-3 hr for tissue of 1-10 mm3. This process does not necessarily fix the entire tissue block. Large tissue blocks will have a core of relatively unfixed tissue, and small blocks will be fixed to a much greater extent. However, the purpose of the fixation step is not to completely fix the tissue but to preserve and harden the tissue sufficiently to improve the tissue morphology from that of unfixed snap-frozen material. Complete fixation is not critical at this point in the protocol, as a second fixation step on the tissue sections is performed just before hybridization and will fix all sections equally. Although tissues can be fixed for periods longer than 3 hr, extended fixation times create additional problems. Overfiition often causes a greater

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PRINCIPLES OF IN SITU HYBRIDIZATION

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Table 1. Tissue adherence t o glass microscope slider prepared with different coatings (see text)"
PLL Acid/PLL NaOH / PLL 0.5% AAS 1.0% AAS 2.0% AAS Maple's Gelatin-chromalum Vectabond 1.8 1.8 2.7 1.8 1.6 1.3 2.0
0

Poly-L-lysine (PU). Slides were washed for 2 min each in dH20 and 100% ethanol twice, followed by drying in an oven at 42'C. The slideswere dipped in 100 pglml poly-clpine (MW 47,000; Sigma) in sterile dH2O for 20 sec. The slides were allowed to dry for 30 min at room temperature, dipped again in the PLL solution, and dried overnight at 42'C. The slides can be stored at room temperature for up to 6 months. NaOHIPLL. This method included initial immersion of the slides in 0.2 N NaOH for 30 min. followed by two dH20 washes, two ethanol washes, and PLL coating as above. Some slides were etched in 1 N NaOH or by boiling in H2S04:HNO3 as outlined in the Maple's procedure (acidlPLL) before PLL coating, but this did not appear to improve tissue retention on the slides. Gelatin-Chrodum Subbing. This was a standard coating technique used in many histology laboratories, consisting of 2.5 g/liter gelatin and 0.25 g/liter chromalum dissolved in dH20 and used to coat ethanol-washed slides. Vectabond. This was used as directed by the manufacturer. Slides were washed for 5 min in acetone, immersed in a Vectabond/acetone solution (1 bottle Vectabond with 350 ml acetone) for 5 min, rinsed for 1 min in dH20 with some agitation, drained, and dried overnight at 42'C. Neoprene. Neoprene (1.5 g) was dissolved in 300 ml isopentyl acetate in a fume hood over a 6-hr period (do not apply heat, as the isopentyl acetate is highly flammable). The slideswere then treated for 5 min in isopentyl acetate, 5 min in 0.5% neoprene-isopentyl acetate, followed by a wash for 30 sec in dH20, and allowed to dry overnight at 37'C before use.

3.0

a At least six slides from two tissues in each experiment were evaluated for tissue retention and morphology after a mock i situ hybridization. The results were n graded as complete tissue loss (O),few fragments of tissue left (I), fair morphology with moderate folding or tissue lass (2), good tissue morphology without tissue loss (3).

the hybridization buffers tend to roll off to one side of the tissue because the gelatin layer wets unevenly. Poly-L-lysine and silane coatings, on the other hand, do not wet, and the high surface tension in the hybridization bubble keeps the buffer in place. As long as the hybridizations are conducted in a humid chamber containing a buffer (4 x SSC, 50% formamide) with a similar vapor pressure as the hybridization buffer, the sections will not dry out during the overnight incubation. Water alone should not be used to maintain the humid atmosphere, as the hybridization solutions are hygroscopic and will take up water during the incubation and dilute the probe. To compare different slide coatings, we have prepared tissue sections from different tissues that in particular tended not to adhere well during the in situ hybridization procedure. These tissue sectionswere then carried through a mock hybridization procedure (no labeled probe added) and the ultimate tissue morphology evaluated after the final washing and drying steps. The following slide coatings were evaluated. Maple's. Slides were acid-washed by boiling at 130C for 10 min in H2S04:HNOs (9:1), then allowed to cool to room temperature and washed thoroughly in'dH2O. The washed slides were then soaked in 2% aqueous solution of triethoxy-3-aminopropyl silane, pH 3, overnight at 65'C, washed in dH20, immersed in 10% glutaraldehyde, pH 6.5, for 60 min. and washed in dH20. Before use the slides were activated by treatment with 0.15 M sodium metaperiodate for 30 min. followed by washing in dH20 and drying. Some slides were coated as above except for substitution of dH20 and ethanol washes similar to that used in the PLL method below for the acid-wash step, with similar results. Amino-acyl Silane (AAS). The slides were washed in dH20, dried in an oven, and dipped for 10-15 sec into asolution of 0.5, 1, or 2 % triethoxy3-aminopropyl silane (Sigma; St Louis, MO) in acetone. The slides were then dried and rinsed in at least three changes of dH20, followed by drying at 50C and storage.

35S-Riboprobe Synthesis (8)

1. Pipet 12.5 p1 [3'S]-UTP(1200 Ci/mmol) into Eppendorf tube. Final concentration should be 12 pM. Dry in speed vac. Do not use nucleotide that has been previously thawed after receipt from manufacturer. Order 250-pCivials and use them all up at once or t r wthe excess away. ho 2. To tube with the dried down probe add 2 p l 5 x Transcription buffer 1 pl IYlT, 100 mM 1 pl FWAsin pl DNA (linearized plasmid 1 @pl) 2 p1 GTP+CTP+ATP mix (stock solution containing 2.5 mM each) 2 pl sterile dH2O Mix thoroughly and centrifuge. Add 1 pl RNA Polymerase (SP6, T7, or T3 as required) Mix gently by pipetting, do not vortex, incubate 1-2 hr, 37%. (All ofthe above buffers are ready-made stocks from Promega Biotech, Madison, WI). 3a. To stop the reaction: Vortex Take out 1 pl of the transcription mixture to determine total incorporation (Step 3b) Add 1 pl RQ1 DNAse to the transcription reaction above Incubate 15 min, 37'C 3b. To monitor incorporation: Eke 1 pl from reaction and place in microfuge tube Add 99 pl 1 x TE Pipet 1 pl of 1:lOO dilution onto a small piece of DE81 paper Wash three times for 5 min in 0.5 M NaP04, pH 7.4 Wash 10 sec in dH20 Rinse briefly (<lo sec) in 100% ETOH Dry thoroughly and count in 10 ml scintillation fluid 4. To extract RNA, after DNAse step add to the reaction: 20 p1 1 x TE 1 p1 tRNA (50 mglml)

Table 2. Companion of Vectabond and Supeq+ost/Plus slides for tissue adherence after in situ hybn2izationa
Superfrost/ Plus Vectabond-coated Superfrost/ Plus Sigma Silane-Prep NaOH/PLL Neoprene Vectabond
a

2.9 2.2
0

2.9 1.9 1.9

Mock hybridizations were conducted as indicated in Table 1 and scored from

0-3 for the amount of tissue left after hybridization.

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1728

-ox

5.

Vortex Add 15 pl phenol, vortex Add 15 p1 CHC13, vortex thoroughly, centrifuge 3 min Remove top (aqueous) fraction carefully with pipette, place in clean microfuge tube Add 15 pl 1 x T to the first tube, vortex, centrifuge 3 min E Remove top fraction, adding it to the first aqueous fraction Add to the aqueous extracts: 55 PI 1 x TE 10 p1 3 M NaAc, pH 7.5 250 pl cold 100% ETOH Keep on dry ice for at least 30 min or store at - 70C up to 1 week Centrifuge 5 min, pour off supernatant (HOT waste) Add 500 pl cold 70% ETOH, vortex Centrifuge 5 min, pour off supernatant (HOT waste) Dry in speed-vac Dissolve pellet in 1 x TE to a final concentration of 300,000 cpm/pl (total determined at Step 3b) Use immediately for in situ hybridization

OR
Store 100-pl aliquots at -7OC up to 1 week as ethanol precipitate with 10 pl 3 M NaAc, pH 7.5, 250 pl cold 100% ETOH 6. Just before starting the hybridization step: Centrifuge 5 min. pour off supernatant (HOT waste) Add 500 p1 cold 70% ETOH, vortex Centrifuge 5 min. pour off supernatant (HOT waste) Dry in speed-vac Dissolve pellet in 100 pl 1 x TE (to a final concentration of 300,000 cpm/d) 7. Make up hybridization mix as described in the in situ methods Probes are labeled by transcription (8), using 35S-labeled UTP (specific An activity >lo00 Ci/mmol) (Amersham; Arlington Heights, E). aliquot of the 3S-labeled nucleotide is pipetted into a microcentrifuge tube and dried under vacuum with centrifugation in a speed-vac (Savant; Farmingdale, NY). transcription reaction is begun by adding in sequential orThe der: 2 PI 5 x transcription buffer (containing 200 mM Tris-HC1. pH 7.5, 30 mM MgC12, 10 mM spermidine, 50 mM NaCl (Promega;Madison, WI). 1 p1 100 mM DTT, 1 pl RNAsin (20-40 U) (Promega), 1 pl linearized plasmid DNA as a template (1 pg/pl), 2 pI of a GCA solution containing 2.5 mM each of GTP, CTP, and ATP in H20, and 2 p1 sterile dH2O. The components are thoroughly mixed to ensure that the labeled nucleotide is in solution and then briefly centrifuged to bring the mixture to the bottom of the tube. The reaction is then started by the addition of 1 pl of the appropriate RNA polymerase (SP6, T7, or T3). depending on the orientation of the cDNA insert or the vector used. The polymerase is added and mixed gently by pipetting without vortexing or centrifugation and the reaction incubated for 60 min at 37C. To increase the amount of probe produced, additional enzyme can be added after 60 min and the reaction allowed to continue for another 60 min. The size of the probes produced should be checked on a 5.2% acrylamide, 7 M urea gel and autoradiography. Although there will be many small bands on the gel, the major band of transcribed material should be equal to the size of the cDNA insert. The protocol presented here does not call for base hydrolysis of the riboprobe before use in hybridization studies. We use full-length (which is really a mixture of sizes) transcripts from probes up to 1.3 KB in length. If the probe is longer than that, it would be preferable to find internal restriction sites for linearization so the resulting template will be less than 1.3KB. Alternatively, short non-overlapping >and3 fragmentsofthe probe could be subcloned into riboprobe vectors. These can be linearized and used individuallyor as a cocktail for transcription and hybridization. Base hydrolysis produces fragments with a wide range of sizes; the smaller frag

menu produced may lack specificity and increase the spurious binding of the probe in the tissue, thus increasing the background. The concentration of 3S-labeled UTP in the transcription reaction should exceed 10 pM without adding any unlabeled material. It is usually more difficult to get full-length transcripts using nucleotide concentrations less than 10 pm, as the labeled nucleotide becomes rate-limiting and the reaction stutters. It is important that the 35S-labeled UTP is used immediately after thawing and that any remaining label should be discarded rather than re-frozenfor use at a later time. We have found that the single greatest contribution to high backgrounds using 3S-labeled probes comes from repeated freezing and thawing of the isotope before use. According to Amersham, there is about a 5 % degradation of the nucleotide with freezing and thawing that might contribute to background problems. It is therefore convenient to use only 250-pCi vials of nucleotide, dividing each vial into two transcription reactions with 125 pCi each. If the specific activity of the isotope is 1200 Ci/mmol and the final volume of the transcription reaction 1 PI, the final concentration will exceed 10 pM. 0 After the transcription reaction, 1 pl of the reaction mixture is removed, diluted 1:1000, and an aliquot pipetted onto a small piece of Whatman DE81 paper to monitor incorporation of the 3S label. The unincorporated nucleotide is washed off the paper with three 5-min washes in 0.5 M sodium phosphate buffer, pH 7.4. The filter paper is rinsed briefly in H20 and ethanol, dried, and counted in the scintillation counter. After removing an aliquot to monitor incorporation of nucleotide, 1 pl of RQ1 DNAse (1 U) (Promega) is added and the reaction allowed to proceed for an additional 15 min at 37C to degrade the template. The probe is then cleaned up by a single phenol-chloroform extraction after the addition of 20 p1 1 x TE (to increase the volume) and 1 pl of tRNA (50mg/ml in dH2O) to act as a carrier, RNAse sink, and to provide a visible pellet in future ethanol precipitations. After the phenol chloroform extraction, the aqueous extract is brought up to 100 pl with the addition of 1 x TE and ethanol-sodium acetate added to precipitate the RNA. After centrifugation, washing in 70% ethanol, and drying, the pellet is resuspended in 1 x TE to a final concentration of 300,000 cpm/pI, using the total incorporation determined above as a guide. Aliquots of the probe (100 111) can then be re-precipitated with ethanol-sodium acetate and the probe stored this way for up to 1 week at - 70C. It is convenient to make multiple aliquots of the probe for a series of hybridizationsover the following week rather than to continuously thaw and refreeze a single aliquot or to prepare probe daily. We have examinedprobe stability over time when stored under ethanol-sodium acetate and have found that there is detectable degradation of the probe size after a week, which could affect the background or hybridization result. Given the relative ease of probe preparation relative to the amount of effort required in the hybridizationprocedure and the time one invests in long autoradiographyexposures,it is preferable to use a freshly transcribed probe for these studies.

Procedure for In Situ Hybridization of Frozen Sections (17-19)

1. Remove slides from freezer, thaw for 5 min at 55C

Fix 10 min in 4% paraformaldehyde-0.1 M NaP04, pH 7.4, 4C 3. Wash 5 min in 0.5 x SSC at room temperature 4. Immerse slides in proteinase K solution, 1-5 pg/ml in RNAse buffer for 10 min at room temperature. The amount of proteinase K must be optimized with each new preparation. Once optimized, aliquots can be frozen and used for some time 5 . Wash for 10 min in 0.5 x SSC at room temperature 6. Pre-hybridization: Dry around sections with Kimwipe, lay slides flat in an air-tight box with a piece of filter paper which has been saturated with Box Buffer (4 x SSC, 50% formamide) on the bottom.
2.

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7.

8.

9. 10. 11.
12.

13. 14. 15. 16.

17.

Cover each section with 100 pl of rHB2 without probe (can use 50 pl if the tissue is small). Incubate at 42C for 1-3 h riboprobc. Assuming that you have Hybridization M k For 35S-labeled used 100 pl of pre-hybridization buffer, combine the following: 2 p1 probe per slide (stock solution 300,000 cpm/pl in 1 x "E); 1 4tRNA per slide (50 mglml stock in sdH2O). Heat 3 min, 95'C. im"Ltei'y add 17 pl ice-cold rHB2 per slide, vortex, place on ice (adjust volumes if you have used less than 100 p1 for pre-hybridization) Hybridization: Add 20 p1 of above hybridization mix to each 100 pl ofpre-hybridization solution directly into the bubble covering the section. Incubate overnight at 55'C (adjust volume if you have used less t a 100 pl for pre-hybridization) hn Wash twice for 10 min in 2 x SSC with 10 mM PME-1 mM EDTA at room temperature (HOT waste) Immerse in RNAse A solution (20 p g / d in RNAse bu&r) 30 min at room temperature (HOT waste) Wash twice for 10 min in 2 x SSC with 10 mM PME-1 mM EDTA at room temperature (HOTwaste) Wash2hrin4litersofO.l x SSCwithlOmMPME-lmMEDTA.55'C Wash twice for 10 min in 0.5 x SSC without PME or EDTA at room temperature Dehydrate 2 min each in 50%, 70%. and 90% ethanol, each containing 0.3 M N&Ac Dry in vacuum desiccator (3-4 hr), store with desiccant u t l autorani diography Dip in Kodak NTB2 nudear emulsion diluted 1:l wt water at 42'C, ih dry for 2 hr in the dark, expose in the dark at 4'C with desiccant for 2-12 weeks Develop at 15'C a. 3 min Kodak Dl9 diluted 1:l with water b. 20 sec in water stop c. 3 min Kodak Fixer, full strength d. Three times for 5 min in water e. Counterstain with hematoxylin and eosin

Once the probe and slides have been prepared it is relatively easy to hybridize as many as 100-200 slides per day. The frozen sections are removed from the freezer and collected on a metal slide tray. The slides are then thawed and dried by incubating them for 5 min exactly at 50C. The drying at this step has been found to improve tissue retention on the slide throughout the hybridization procedure. After drying, the slides are put into plastic slide carriers (each holding 25 slides) and immersed in 4% paraformaldehyde for 10 min at 4'C to ensure that the tissue is equally fixed. This fixation also tends to improve tissue retention on the slide. After fixation the slides are washed in 0.5 x SSC (1 x SSC = 150mM NaCI, 15 mM sodium citrate, pH 7) and permeabilized in 1 pg/ml proteinase K (Sigma)in 500 mM NaCI, 10 mM Tris, pH 8, for 10 min at room temperature. This is important, as permeabilization with proteinase K, pronase, or HCI has been shown to improve the signal intensity after in situ hybridization, presumably because they either loosen up the membranes and allow greater penetration of the probes or because they partly digest proteins associated with the RNA, allowing longer regions access to the probes (lJ6). The amount of proteinase K must be optimized for each lot used. It is suggested that this should be tested by doing a mock hybridization with a range of proteinase K concentrations before starting actual experiments. A good starting point is that concentration of proteinase K which provides the best final tissue morphology and tissue retention on the slides at the end of the mock hybridization procedure. Once a hybridization signal is obtained, it would be appropriate to go back and then optimize the signal by again testing various concentrationsof proteinase K. One of the biggest problems with the technique, as mentioned earlier, is keeping the sections on the slide throughout the hybridization procedure. If this becomes a serious problem, the first thing to do is eliminate the proteinase K step. It

is important to remember that although proteinase K improves the signal intensity, it is not necessary to get a signal in the first place. After the proteinase K treatment the slides are washed for 10 min in 0.5 x SSC, dried carefully around the section with a lint-free tissue, and placed in air-tight hybridization boxes (Nalgene utility boxes, Baxter Health Care ProdbctsUL.1995-4) containing filter paper saturated with Box Buffer (4 x SSC, 50% formamide). The pre-hybridization is begun by pipetting a small amount of hybridization buffer (100 pl) onto the sections (for riboprobes rHB2: 10 mM DTT, 0.3 M NaCI. 20 mM Tris, pH 8 , 5 mMEDTA, 1 x Denhardt's, 10% dextran sulfate, 50% formamide;for oligomers HB8: 10 mM DM, 1 x Denhardt's, 5 x SSC, 100 pg/ml ssDNA, 100 pg/ml tRNA, 10% dextran sulfate, 20% formamide).The tissucS are pre-hybridized for 1-3 hr at 42C and then the hybridization is begun by adding 20 pl of a probe mixture which is pipetted carefully into the bubble of prehybridization solution covering the tissue section. The hybridization mixture contains 2 pl 3sS-labeledriboprobe (300,000 cpm/pI in 1 x E), 1 p1 tRNA (50 mg/ml stock in dH2O), and 17 pl rHB2 for each 100 p1 of pre-hybridizationbuffer on the slides. This is prepared by adding the probe and tRNA to a microfuge tube and heating this at 95'C for 3 min in a heat block, followed by the immediate addition of ice-cold rHB2. The solution is then vortexed and kept on ice until used for the hybridization. Once the probe mix is added to the slides, the boxes are covered again and placed in a 55'C incubator overnight. Coverslippingof the slides is not necessary, nor is it recommended during the hybridization incubation. The surface tension of the hybridization buffer is sufficient to keep the probe mixture in contact with the tissue overnight, and as long as the hybridization is carried out in sealed boxes with filter paper saturated with 4 x SSC, 50% formamide, the tissue sections will not dry out. Coverslippinghas its own problems with air bubbles and potential damage to the tissue or radioactive contamination of the lab on removal of the coverslips after the hybridization is complete. Extreme care should be taken to ensure that all buffers, reagents, slide holders are ribonuclease (RNAse) free up to and including the hybridization at Step 8 . RNAse contamination is not a great problem in the posthybridization steps with riboprobes, since the tissues will be treated with RNAse as part of the stringency wash. For this reason, care should be taken that all of the slide holders and beakers used after Step 8 are kept for that purpose alone and never used during the pre-hybridization procedure, as these materials will probably remain contaminated with RNAse. It is sug gested that all equipment and containers used for the pre-hybridization steps be periodically treated with diethyl pyrocarbonate (DEPC; Sigma). This is done by soaking the equipment in fresh 0.1% solution of DEPC overnight in the fume hood. The DEPC should be diluted in sterile dH20 and used immediately, as it degrades quickly. The hybridization reaction is essentially complete after 4 hr but it is convenient to let the incubation go overnight before the post-hybridization washes are performed. The slides are removed from the hybridization boxes and rinsed twice for 10 min each in 2 x SSC containing 10 mM p-mer. captoethanol and 1 mM EDTA (PME/EDTA) at room temperature to wash off the majority of radioactive probe associated with the slides. This is followed by RNAse A treatment (Sigma) (20 pg/ml in 500 mM NaCI, 10 mM Tris, pH 8) for 30 min at room temperature. RNAse A will attack singlestranded RNA but will spare RNA-RNA duplexes, thus ensuring that the probe is properly complexed to the mRNA by complementary base pairing. RNAse treatment will significantly reduce the specific signal but will help to provide the extremely low backgrounds that enable the detection of very low copy number mRNAs in tissue using this technique. Great care must be taken with the handling of the RNase at this stage to prevent any cross-contaminationof pipettes or equipment, which will cause problems with later hybridizations. For this reason, all equipment used in the post-hybridization washing steps is kept completely separate from material used for the pre-hybridization steps. The RNAse treatment is followed by two more 2 x SSC PMEIEDTA washes at room temperature

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and then a high-stringency wash in 4 liters of 0.1 x SSC BMElEDTA at 55'C for 2 hr. This can be done in a large 4-liter beaker on a stir plate heated with an immersion heater. This is followed by two washes with 0.5 x SSC without BMElEDTA at room temperature. The PMEIEDTA is added to keep the 3'S-labeled probe in a reduced state throughout the washing steps. which may reduce backgrounds. It is important. however, to remove the PMElEDTA before coating the sections with photographic emulsion, as reducing agents would cause chemical reduction of the silver salts and high background. Finally, the tissue sections are dehydrated in graded alcohols containing 0.3 M NH4Ac. dried in a vacuum desiccator, and can be stored desiccated at room temperature until coating with photographic emulsion. The slides are exposed for various lengths of time in the dark in sealed slide boxes with desiccant at 4'C. The exposure time may vary depending on the amount of mRNA in the tissue. This can range from 3 days for an abundant mRNA (pro-opiomelanocortin in the rat pituitary) (16) to 8 weeks for platelet-derived growth factor expression in human vascular tissue (19) or even 12 weeks for rare mRNAs such as tumor necrosis factor or tissue plasminogen activator ( 5 ) . Because it is not always possible to know the optimal exposure time for each tissue or probe, it is a good idea to perform all hybridizations in triplicate and develop one slide every 4 weeks until the optimal exposure is determined.

Modifcutions for Purufin Sections or Cultured Cell's

The same basic protocol described in the preceding sections can also be used for paraffin sections or cultured cells, with slight modifications. Paraffin embedding after paraformaldehyde fixation does provide good preservation of the cellular mRNA for hybridization. Direct comparison of paraffin to frozen blocks from the same tissue indicates that there is approximately a 25% loss in hybridization signal due to paraffin (Smith and Wilcox. unpublished observations). Therefore, when paraffin embedding is necessary for appropriate tissue morphology (i.e.. lung), then it can be used. Sections are deparaffinized by washing twice for 2 min in xylene, twice in 100% ethanol for 1 min. for 1 min each in 95% ethanol, 70% ethanol. 50% ethanol, and 0.5 x SSC. The sections are then ready for hybridization in the above protocol, beginning with the paraformaldehyde fixation at Step 2. In the same manner, cultured cells can be fixed and prepared for hybridization. Cells are either grown on Lab-Tek culture slides or centrifuged onto Vectabond-coated or SuperfrostlPlus slides and then fixed by immersion in 4% paraformaldehyde at 4'C for 10 min. The slides are then stored at 4'C for up to 3 months in 70% ethanol. Before hybridization the slides are taken from the ethanol and the hybridization procedure is begun by immersing them in 0.5 x SSC. starting with Step 3 directly.

Control's
The choice of an appropriate control is the most challenging aspect to using in situ hybridization properly. A number of controls have been used by investigators to establish the validity of their results (see below): some are better than others. The best control is common sense. Does the signal make sense to you? Is it localized over the cytoplasm of the cell rather than over the nucleus? Are there negative cells in the tissue as well as positive cells? Is the gene expressed in appropriate sites in control tissues? These are important questions that must be addressed each time the results of an in situ experiment are evaluated. It is easy to assume that any concentration of silver grains represents a positive signal but careful examination and rehybridization may yield very different results (Figure 1).

Figure 1. Examples of a good (A) and bad (B) in situ hybridization result. Sections of rat pituitary were hybridized with 35S-labeled pro-optomelanocortin (POMC)riboprobes using two different methods. (A) Section hybridized according to the protocol outlined in this review reveals strong hybridization to the intermediate lobe (central band of positive cells), scattered hybridization to anterior lobe corticotrophs (to the left), with no hybridization to the posterior lobe (to the right of the intermediate lobe). (B) The same pituitary hybridized in parallel with the section in A using a riboprobe procedure outlined in a recent series of publications (6,11,13). Note the high background in the posterior lobe in B and the lack o any significant hybridization signal in the anterior lobe. In f the absence of any additional information, the background in B may have been assumed to represent positive hybridization and illustrates the problem associated with accepting all hybridization results at face value. Exposure 1 week; photographed with polarized light epiluminesence (Leitz). Original magnification x 50. Bar = 100 pm.

Controls for In Situ Hybridization SenseIAntisense CO-localization with protein Multiple non-overlapping probes Northern blots Multiple probes RNAselDNAse Common Sense Sense and anti-sense hybridizations are widely used as controls for in situ hybridizations with riboprobes (Figure 2). Riboprobes are synthesized by transcription utilizing the cDNA as a template for probe synthesis. Mes-

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3
5
ECoRl

GGTCCA CCAGGT

DNA
3 BamHl

u3,
SenseA CRiboprobe ~GG C 5
5

CCAGGU mRNA

GG~CCA

CCAGGU mRNA
9

IIIIII

Antisense Riboprobe probe

Figure 2. Transcription of sense and anti-sense riboprobesfor use as controls for in situ hybridization. Cellular mRNA is transcribed by RNA polymerase in the 3to 5direction using one strand of the chromosomal DNA as a template. Dependingon the orientation of the cDNA insert in a riboprobe vector relative to the SP6 or T7 RNA polymerase initiation sites, either sense or anti-sense RNA probes can be synthesized. Transcription from the CDNA template using SP6 RNA polymerase in this example produces a nucleotide sequence identical to the mRNA, otherwise known as the sense transcript. Since the sense probe is the same sequence as the cellular mRNA, it will not bind during the hybridization reaction and can be used as a negative control. Transcription of the cDNA template from the opposite end, using the T7 RNA polymerase initiao tion site, will produce an anti-senseRNA probewhich will hybridize t the mRNA in tissues.Anti-sense transcriptswill be generatedwhen the 3end of the cDNA is closest to the site of transcription initiation.

senger RNA is normally synthesized from chromosomal DNA in the 3 to 5 direction, producing sense mRNA. To make an anti-senseriboprobe the cDNA insert is subcloned in a transcription vector in the 3 to 5 direction relative to an RNA polymerase initiation site. Transcription takes place in the presence of a labeled nucleotide so that the resulting anti-sense probe is complementary to the mRNA and will bind to it during the hybridization reaction. Insertion of the cDNA insert in the opposite orientation relative to the RNA polymerase initiation site will transcribe the control sense riboprobe. a sequence identical to the mRNA in the tissues which will not hybridize. Since the sense and anti-sense probes both contain the same relative proportion of all of the nucleotides (A/Us or G I G ) in the same sequence, this has been consideredan excellent controlfor riboprobehybridizations (2).

One word of caution is warranted regarding the use of sense and antisense riboprobes. It is often possible to see positive hybridization or eveh high backgrounds with sense riboprobes yet to find a very strong, specific and reproducible signal in the parallel anti-sensehybridization. H w should o this be interpreted? Is there anti-sense mRNA in the tissue? Is the enure hybridization flawed?The first assumption is that the researcher has done something wrong and the experiment is usually repeated until the sense hybridizations are appropriatelynegative and the anti-sense hybridizations appropriately positive. However, the question remains: was the first or second experiment flawed? There is no easy answer to these questions. and the assumption is that sense hybridizations should be negative. A common approach used in my laboratory is hybridization with multiple probes in a single experiment. This can ensure specificity and provides the maximal amount of information per experiment. Typically, four anti-sense probes are hybridized in a given experiment and the results of each hybridization compared for specificity.The probes should show more than one hybridization pattern for the experiment to succeed and should include at least one probe that will hybridize to a known subset of cells in the sections being examined. A good control for human tissue is von Willebrands Factor (VWF), which is synthesizedonly by endothelial cells. Other cell types in the tissue will be, or should be, negative, and the researcher can evaluate tissue background with this approach. The use of a positive control probe with a known cellular distribution of hybridization in the tissues also provides a control for positive mRNA hybridization and the efficiency of the experiment. The specificity of the hybridization is indicated when one probe, e.g., VWF, hybridizes to endothelial cells and another probe hybridizes to other cells in serial sections (19). This is a good control and is generally accepted by journal reviewers. The mistake people commonly make with this approach is the choice of the control probe. Often tissues are hybridized with poly d(T) to detect poly(A) mRNA tails or with housekeeping genes such as actin, known to be expressed by all cells. These are poor choices for controls, as the result will be positive hybridization to all cells with no negative cells present to determine the amount of background hybridization. The biggest problem with in situ hybridization is background and determining when accumulation of silver grains is real ar not. For this reason it is important to have clear positive and negative hybridizations included in an experimentso that it is clear what each will look like. Hybridization of cDNA or riboprobes to tissue sections obviously depends on the presence of mRNA in the tissues. For t i reason, another hs control that has been used is predigestion ofthe tissue sectionswith RNAse and DNAse. Since RNAse, but not DNAse, should degrade the signal, this is assumed to confirm the necessity of cellular mRNA for the hybridization reaction. One problem with this approach is its application to hybridization with riboprobes and the possible degradation of the probe itself rather than the tissue A. Additional controls that have been used include many that fall in the common sense category. Multiple non-overlappingprobes encoding the mPNA should each give similar hybridization signals, whereas an irrelevant nucleotide sequence should not. This is an especially useful control when synthetic oligomers are used for hybridization and it is possible to make a number of different probes. Alternatively, non-overlapping5 and 3 ends of the cDNA can be subcloned and used for hybridization. Depending on the level ofgene expression in the tissue it should be possible to detect the mRNA by Northern blots. This would at least confirm the presence of mRNA in the tissues under study. Of course, this assumes that there is enough tissue for a Northern blot or enough cells expressing the gene so that the mRNA can be detected with this technique. Alternatively, co-localizationof the protein and mRNA using immunohistochemistryand in situ hybridization on serial 01 same sections can provide another level of assurance that the m situ hybridization signal is real. When exon-specific probes are used, the in situ hybridization signal

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as to use the number of grains over the nucleus to quantify the amount of mRNA associated with the cell (11.13). The mRNA signal with exonspecific probes should be over the cytoplasm (Figure 3). There will be some spread of grains over the nucleus, but 90% or more of the signal normally appears as a cluster of silver grains around the nucleus of the cell. Depending on the cell morphology, it is possible to see some nuclear silver grains. For example, monocytes or small macrophages with a very thin cytoplasm often show some grains over the nucleus (10). This is probably because the mRNA is concentrated in a restricted cytoplasm of which the major part appearing in a tissue cross-section overlies the nucleus. On the other hand, one of the most common background problems is nuclear silver grains. When the hybridization is pushed by increasing the probe concentration too high or by dropping the stringency of the final wash. the first thing that happens is that all of the cells in the section show a nuclear signal. This does not mean that the mRNA is in every cell but rather that it is time to begin to trouble-shoot the experiment to determine the source of background.

Summary
In situ hybridization provides the researcher with 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. It is a difficult procedure, and one must ask whether it is worthwhile to develop it for a single experiment or whether a Northern blot or some other assay might provide the desired information with a lot less effort. I have tried to provide a starting point for those who wish to begin using in situ hybridization in their own laboratories. The procedure outlined here 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 background in the tissue. However, if this procedure is applied correctly, background is not a problem. A good hybridization will have 25-50 cytoplasmic (not nuclear) grains per positive cell and <1 grain per negative cell or cell-sized area of tissue matrix. The improvement in the signal-to-signal noise ratio comes from the use of ribonuclease and high-stringencywashes in the post-hybridization steps. Although these washes do reduce the specific signal, they enhance the ultimate level of detection of low copy number As 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. Although 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

Figure 3. Localization of the in situ hybridization signal in the cytoplasm with exon-specific probes. Rat pituitary sections were hybridized with (A) 3H- or (B) biotin-labeled riboprobes directed against exon 3 of the POMC cDNA. POMC riboprobes were transcribed using 3H-labeled UTP and CTP (10 pM each) in the transcription reaction, hybridized according to the protocol outlined in this review, and exposed to photographic emulsion for 5 weeks. Biotin-labeled riboprobeswere transcribed using biotinylated UTP and the BRL non-radioactive RNA labeling system kit (Gibco BRL; Gaithersburg, MD) and hybridized using the BRL in situ hybridization and detection system kit exactly as described by the manufacturer. Although hybridizations with 3H-labeled or biotin-labeled riboprobesare not as sensitive as 35S-labeledprobes, they can provide excellent resolution of the mRNA signal. Note the specific cytoplasmicsignal using both methods and the lack of any nuclear binding of the probes. Original magnifications: A x 200; B x 100. Bars = 100 pm.

should be found primarily over the cytoplasm of the cell (3.4). This is in contrast to recently published reports examining local gene expression in biopsy specimens of human coronary arteries (6.11.13). These authors accepted nuclear grains as indicating a positive hybridization and go so far

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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 ensure that the signal is real and not due to some artifactual binding of the probe to the tissue.

SM: PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol 111:2149, 1990 8. Melton DA, Krieg PA, Rebagliati MR, 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. Nucleic Acids Res 1237035, 1984 9. Nelkin NA. Coughlin SR, Gordon D, WilcoxJN: Monocyte chemoattractant protein-1 in human atheromatousplaques.J Clin Invest 881121, 1991 10. Neken NA, Soifer SJ, OkeefeJ, Vu TK, Char0 IF, Coughlin SR Thrombin receptor expression in normal and atherosclerotichuman arteries. J Clin Invest 901614, 1992 11. Nikol S, IsnerJM, PickeringJG, Kearney M, Leclerc G, Weir L: Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions. J Clin Invest 90:1582, 1992 12. Rosenthal A. Chan SY, Henzel W, Haskell C, Kuang WJ, ChenE, 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, 1987 13. Simons M, Leclerc G, Safian RD, IsnerJM, Weir L, Baim DS: Relation between activated smooth-muscle cells in coronary-artery lesions and restenosis after atherectomy. N Engl J Med 328:608, 1993 14. WilcoxJN: Analysis of local gene expression in human atherosclerotic plaques. J Vasc Surg 15:913, 1992 15. Wilcox JN, Derynck R Developmental expression of transforming growth factors alpha and beta in m o w fetus. Mol Cell Biol8:3415, 1988 16. WilcoxJN, Gee CE, Roberts J L In situ cDNA:mRNA hybridization: development of a technique to measure mRNA levels in individual cells. Methods Enzymol 124510, 1986 17. WilcoxJN, Pollard H, Moreau J, SchwartzJC, Malfroy B: Localization of enkephalinasemRNA in rat brain by in situ hybridization: comparison with immunohistochemicallocalizationof the protein. Neuropep tides 1477, 1989 18. WilcoxJN, Smith KM, Schwartz SM, Gordon D: Localizationof tissue factor in the normal vessel wall and in the atheroscleroticplaque. Proc Natl Acad Sci USA 86:2839, 1989 19. WilcoxJN, Smith KM, Williams I , T Schwartz SM, Gordon D: Plateletderived growth factor mRNA detection in human atheroscleroticplaques by in situ hybridization. J Clin Invest 82:1134, 1988

Acknowledgments
I am deep/y indebted to a number ofpeople who have worked with me over the past 10 years, each of whom bas contributed to the development of this in situ protocol in some way, including Kathy Smith,Judy Hasko, Andrew Augustine, and Romesh Subramanian. I am especial/y indebted to Jose Rodriguezfor providing the in situ hybridizationspresented here.

Literature Cited
1. Armstrong E, PartanenJ, Cannizzaro L, Huebner K, Alitalo K Localization of the fibroblast growth factor receptor-4 gene to chromosome region 5q33-qter. Genes Chromosom Cancer 494, 1992
2. Cox KH, DeLeon DV, Angerer LM, Angerer RC Detection of As

in sea urchin embryos by in situ hybridization using asymmetricRNA probes. Dev Biol 101:485, 1984 3. Fremeau RT, Autelitano DJ, Blum M, WilcoxJ, RobertsJ L Intervening sequence-specific in situ hybridization: detection of the proopiomelanocortingene primary transcript in individual neurons. Brain Res Mol Brain Res 6:197, 1989 4. Fremeau RT, LundbladJR, Pritchett DB, WilcoxJN, RobertsJL Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei. Science 234:1265, 1986 5. Gordon D, Augustine AJ, Smith KM, Schwartz SM, WilcoxJ N Localization of cells expressing tPA, PAIL and urokinase by in situ hybridization in human atherosclerotic plaques and in the normal rhesus monkey. Thromb Haemost 62:131, 1989 6. Leclerc G, IsnerJM, Kearney M, Simons M, Safian RD, Baim DS, Weir L Evidence implicating nonmuscle myosin in restenosis. Use of in situ hybridization CO analyze human d a r lesions obtained by directional atherectomy. Circulation 85:543, 1992 7. Majesky MW, Reidy MA,Bowen Fbpe DF, Hart CE, WilcoxJN, Schwartz

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