In situ hybridization: Quantitation using radiolabeled hybridization probes

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[53] I n S i t u H y b r i d i z a t i o n : Q u a n t i t a t i o n U s i n g R a d i o l a b e l e d Hybridization Probes By GEORGE R. UHL

Introduction In situ hybridization techniques are finding increasing use when localization of gene expression is of importance, and especially when regulated gene expression is studied in heterogeneous cell populations. Recent reviews document approaches to localizing specific nucleic acid sequences to particular cell populations by in situ hybridization. ~-5 Here, we explore some of the issues and caveats involved when the techniques are used in a quantitated fashion. How can in situ hybridization yield data that can be interpreted quantitatively? What are the limitations of interpretation of these data? Studies of the behavior of individual cells in regulating gene expression provide impetus for these quantitative concerns. In the brain, for example, large numbers of genes are expressed, but the expression of many of these important genes is highly localized to discrete cellular subpopulations, t,6 Thus, studies of nucleic acids extracted from whole tissue can fail to elucidate features of gene regulation that may have great functional significance. If the differential distribution of gene expression from one cell type to another is important, or if regulated changes in levels of cellular expression are of interest, quantitated in situ hybridization techniques are the approaches of choice. Two sorts of information can be sought in these studies. First, comparisons of the cellular expression of specific nucleic acid sequences in two particular states often yield biologically important information. These questions most frequently concern regulation of the expression of specific messenger RNAs; we focus on these mRNAs as targets of hybridization. In these settings, relative determinations of the cellular levels of hybridiz-

t G. R. Uhl, ed., "In Situ Hybridization in Brain." Plenum, New York, 1986. 2 B. D. Shivers, B. S. Schachter, and D. W. Pfaff, this series, Vol. 124, p. 497. 3 j. N. Wilcox, C. E. Gee, and J. L. Roberts, this series, Vol. 124, p. 510. 4 j. D. Penschow, J. Haralambidis, P. Aldred, G. W. Tregear, and J. P. Coghlan, this series, Vol. 124, p. 534. 5 K. Valentino, J. Eberwine, and J. Barchas, "ln-Situ Hybridization: Neurobiological Applications." Raven, New York, 1987. 6 D. M. Chikaraishi, S. S. Deeb, and N. Sueko, Cell (Cambridge, Mass.)13, 111 (1978).

METHODS IN ENZYMOLOGY, VOL. 168

Copyright © 1989 by Academic Press, Inc. AlL rights of reproduction in any form reserved.

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able mRNAs are adequate to address many biological questions. In this chapter, we describe several approaches to determination of relative cellular hybridization densities using in situ hybridization. In other circumstances we might wish to determine the absolute cellular content of mRNA. As we shall see below, however, accurate determination of this second quantity may be more difficult without the use of additional adjunctive studies of extracted RNAs.

Issues in Quantitation of in Situ Hybridization: Classic Kinetics and in Situ Hybridization Classic descriptions of nucleic acid association kinetics emphasize hybridization in solution. 7,8 Hybridization occurs in two phases. In an initial nucleation event, a relatively short nucleic acid sequence recognizes a complementary sequence. This initial recognition aligns stretches of similar sequence and allows subsequent rapid "zippering" of longer complementary sequences. The affinity of hybridization of one nucleic acid to another is classically described in terms of concentration- and time-dependent association parameters (e.g., Cot). Hybridization affinity can also be determined by study of hybridization stringency: the temperature, salt, and formamide conditions that can prevent hybridization or "melt" already formed hybrids. Three characteristics of the target of hybridization in in situ studies, however, combine to produce cautions about using this Cot modeling to exactly describe the results of these studies. First, the target of hybridization is immobilized. This feature invalidates one of the assumptions of classic nucleic acid association kinetics, that each species is free to diffuse toward the other. Second, the hybridization target lies behind a diffusion barrier. The cellular constituents located between the target and the externally applied labeled hybridization probe can serve to "sieve" heterogeneously sized probe molecules, to delay their access, and to provide a substrate for degradation of the probe. These elements can thus induce both qualitative and quantitative changes in hybridization, in comparison to solution hybridization. Third, messenger RNA in vivo is not necessarily accessible for hybridization. Polysomes, nascent polypeptide 7 R. J. Britten and E. H. Davidson, in "Nucleic Acid Hybridization. A Practical Approach" (B. D. Haines and S. J. Higgins, eds.), p. 3.1RL Press, Washington, D.C., 1985. s D. C. Campbell, in "In Situ Hybridization in Brain" (G. R. Uhl, ed.), p. 239. Plenum, New York, 1986.

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chains, and other cellular constituents fixed in apposition to the RNA could provide substantial steric hindrance for hybridization. These factors could result in qualitative changes in the stringency of hybridization (based on interrupting part of the hybridizing sequence) and could also yield quantitative changes in the total amount of hybridization. Each of these features raises concerns about direct application of simple kinetic analyses to in situ hybridization. Fortunately, several empiric approaches can validate in situ quantitation. First, studies in a growing number of biological systems reveal that in situ hybridization signals do vary in a fashion that makes biological sense and that correlates with studies of mRNAs extracted from the same tissues (e.g., Refs. 9-19). When such hybridization differences are found, they can be further validated through attention to features of the hybridization that can influence quantitative results, care with autoradiographic quantitation, and use of either of two tools to approach "absolute" quantitation. In saturation analyses, hybridization responses to increasing probe concentrations are assessed. 2°,21 This can yield interpretable results in relatively uncomplicated test situations. Alternatively, use of mRNA-sense standards can define the relationship between hybridization densities and the concentration of hybridizable mRNA target molecules (J. Palacios, personal communication). 22 We discuss methodologic considerations related to quantitation in the order that they occur in practice: those related to probe, hybridization and standardization, autoradiography, and analysis.

9 G. R. Uhl, H. H. Zingg, and J. F. Habener, Proc. Natl. Acad. Sci. U.S.A. 82, 5555 (1985). to j. M. Rothfield, J. F. Hejtmancik, P. M. Corm, and D. W. Pfaff, Exp. Brain Res. 67, 113 (1987). 1~ B. Wolfson, R. W. Manning, L. G. Davis, R. Arentzen, and F. Baldino, Jr., Nature (London) 315, 59 (1985). n R. T. Fremeau, Jr., J. R. Lundblad, D. B. Pritchett, J. N. Wilcox, and J. L. Roberts, Science 234, 1265 (1986). 13 L. G. Davis, R. Arentzen, J. M. Reid, R. W. Manning, B. Wolfson, K. L. Lawrence, and F. Baldino, Jr., Proc. Natl. Acad. Sci. U.S.A. 83, 1145 (1986). 14 W. S. Young III, E. Mezey, and R. E. Siegel, Neurosci. Lett. 70, 198 (1986). 15 S. A. Lewis and N. J. Cowan, J. Neurochem. 45, 913 (1985). 16 S. M. Reppert and G. R. Uhl, Endocrinology 120, 2483 (1987). 17 G. R. Uhl and S. M. Reppert, Science 232, 390 (1986). t8 B. D. Shivers, R. E. Harlan, G. J. Romano, R. D. Howells, and D. W. Pfaff, in "In Situ Hybridization in Brain" (G. R. Uhl, ed.), p. 3. Plenum, New York, 1986. 19 S. L. Lightman and W. S. Young III, Nature (London) 328, 643 (1987). 20 j. E. Kelsey, S. J. Watson, S. Burke, H. Akil, and J. L. Roberts, J. Neurosci. 6, 38 (1986). 21 j. B. Lawrence and R. H. Singer, Nucleic Acids Res. 13, 1777 (1985). 22 G. R. Uhl, B. Navia, and J. Douglas, J. Neurosci., in press (1988).

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Approaches to Quantitation: Practical Considerations When Hybridizing Probes

An ideal probe for quantitated in situ hybridization studies should have several characteristics that can facilitate interpretation of results. 23 If the probe has a known specific activity and if it is stable under conditions of hybridization, then the amount of radioactivity detected in the tissue can be simply related back to the mass of hybridized probe. Substantial degradation of probe molecules during the in situ hybridization procedures increases uncertainty about the specific activity of the hybridized molecules. In addition, the probe should have relatively ready access to the RNA species targeted. If tissue constituents exert substantial "sieving," hybridization of smaller probe fragments may be favored. Even if these fragments represent a small fraction of the total probe applied to tissue, they might in fact be selected during hybridization. Specific activity of the material in the tissue, then, would be substantially different from that of the starting materials. These considerations are of special concern for nick-translated probes, frequently used as a mixture of different species, and for cRNA probes used after basic hydrolyses or under hybridization conditions that allow breakdown of these easily degraded molecules. Single Probe Concentration Rationale. In instances where the biological question to be addressed concerns the relative amount of hybridizable mRNA in one state compared to another, a single concentration of radiolabeled probe is frequently utilized. These studies have successfully documented changes in the cellular and regional expression of specific mRNAs induced by a large number of stimuli. 9-19 The approach has empiric validation: Under the conditions employed, variation in hybridization densities do correlate with variation in the amount of mRNA that can be extracted from tissue. Further means to buttress the validity of this approach involve use of mRNA-sense RNA or DNA standards, procedures that we and others have recently developed (J. Palacios, personal communication). 22 Using SP6/T7 vector systems, mRNA-sense RNA corresponding to the gene of interest can be synthesized, quantitated spectrophotometrically, diluted appropriately, and applied in known amounts to specific regions of nylon filters or other suitable supports. Alternatively, known amounts of this 23 G. R. Uhl, in "In Situ Hybridization in Brain" (G. R. Uhl, ed.), p. 227. Plenum, New York, 1986.

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RNA or mRNA-sense DNA oligonucleotides can be mixed with brain paste or embedding medium, frozen, and sectioned at the same thickness as the experimental material. These standards can be hybridized in parallel with tissue sections, under the same conditions. The ability of increasing amounts of RNA to provide an increasing hybridization signal and the relationship between the increasing amounts of RNA and increasing hybridization signal can both be assessed. If these interactions are not linear, they can provide estimates that, for example, 2-fold changes in hybridization density may reflect 3-fold changes in mRNA content. m R N A - S e n s e Standards. We construct mRNA-sense standards using cDNAs cloned into plasmids such as pGEM (Promega) that also contain promoters for the active RNA polymerases SP6 and T7. When these plasmids are cut at the end of the cDNA, transcription with the appropriate RNA polymerase yields full-length RNA, as assessed by Northern analyses. RNA concentrations can be determined by absorbance at 260 nm, z4 and a range of dilutions set up. One-microliter aliquots of these RNA solutions can be heated to 65° and spotted onto 1 × I cm nylon filter squares (Nytran, Schleicher and Schuell). Application of a stream of warm air during spotting results in more uniform distribution of the RNA. Under these circumstances, 8-12 standard spots can easily be applied to each filter square. The filters are baked for 2 hr at 80° to fix the RNA to the filter, after which they can be stored at -70 °. Before use, the filter squares can be glued to slides with super-glue. They are then hybridized, washed, and subjected to film autoradiography under the same condition used for experimental tissue sections. mRNA-sense oligonucleotide DNA standards can also be synthesized chemically, quantitated spectrophotometrically, and used for the same purposes, z5 These oligonucleotides may be too short to adhere effectively to nylon or nitrocellulose filters, but they can be embedded in mounting medium or carefully prepared brain paste at known concentrations. Frozen sections of these standards can be thaw-mounted onto slides and hybridized in parallel with the experimental unknown sections. Loss of the standardizing mRNA sense oligonucleotide from the matrix by elution during hybridization and washing can be reduced by fixation of the standards prior to hybridization (J. Palacios, personal communication). These strategies can define the relationship between the density of applied mRNA-sense standardizing material and the accessible mRNA in tissue. They cannot account as easily for variable mRNA retention by standards or variable access to the mRNA in unknown tissue standards. 24 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 55 M. J. Gait, "Oligonucleotide Synthesis." IRL Press, Oxford, 1984.

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Multiple Probe Concentrations Limitations of a Single Probe Concentration. Use of a single concentration of hybridizing probe could have several limitations. If, under the conditions utilized, virtually all of the probe molecules were hybridized to target, then no change in hybridization signal would be seen with increasing concentrations of target mRNA. The use of mRNA-sense standards can provide one control for this feature, if the hybridization densities for the standards encompass the range of hybridization values found in the unknown samples. In addition, it is conceivable that different physiological states examined could be accompanied by differences in accessibility of the target RNA to probe. Use of several different probe concentrations could help to elucidate such differences. Differences both in the barrier to diffusion from hybridization solution to the tissue and in the extent of mRNA obstruction by riboproteins could be overcome in part by increasing probe concentration. Conversely, if a hybridization difference between experimental and control tissues is noted at several different probe concentrations, there is greater assurance that it relates to bona fide differences in the number of molecules of hybridizable target mRNA species. Saturation Analyses. Quantitation of the number of hybridizable mRNA molecules present may be approached using a single probe concentration in conjunction with mRNA-sense standards as noted above. Another approach to determine the maximal number of hybridizable molecules involves saturation analyses, z°,z~ With addition of increasing amounts of radiolabeled probe, hybridization values typically increase up to a point, and then plateau. At this plateau, each accessible molecule of target mRNA is presumed to be hybridized; determination of this plateau hybridization level can then lead to an estimate of the density of the target mRNA. In practice, these studies require relatively large amounts of hybridization probe. They are most readily performed in cultured cells or homogeneous cell populations, where radioactivity can be effectively detected by liquid scintillation counting over a broad range of values. 2°,~1 Autoradiographic quantitation of these results requires multiple exposures, in conjunction with careful autoradiographic standardization (Uhl and Parta, unpublished observations, 1985). Generation of these sorts of data at the level of individual scattered cells requires a substantial graincounting effort. Use of the high probe concentrations necessary to obtain saturation can frequently raise the fraction of probe that is nonspecifically associated with tissue, decrease signal-to-noise ratios, and increase the variability of estimates of the true saturation value.

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Approaches to Quantitation: Quantitation of Radioactivity In simple systems, it may be possible to model features of in situ hybridization reactions by detecting radioactivity through scintillation counting as noted above. 26 In most circumstances, however, the requirement for high anatomic resolution demands utilization of autoradiography. Quantitation of the results of these techniques in turn requires careful attention to specific technical concerns. Autoradiographic methods can yield film or emulsion autoradiograms, but virtually all of the same basic issues pertain to each approach. Each method determines the density of radioactivity over a specific area, which can be related back to the density of hybridized probe and even to the effective concentration of hybridizable mRNA, as noted above. Because of difficulties with anatomic scattering of the emissions from high-energy isotopes and with quenching of low-energy particles, absolute quantitation of the number of molecules in a certain area may be difficult, as noted below. Saturation Each autoradiographic system has only a finite capacity to display the consequences of radioactive decay. Saturation occurs when most of the silver grains available are converted to a latent image; at this point the image can get no blacker. 26-29If two regions in the same experiment both show emulsion saturation, it is still possible that the amounts of radioactivity in each may be different. Thus, it is important to demonstrate that the radioactivity from each of two unknown samples falls within but not at the top of a range of possible values using autoradiographic standards before concluding that the radioactivity emitted by each is reliably sampled autoradiographically. Otherwise, the same values can be attributed to samples that are not in fact equally radioactive, Determining the Operating Characteristic The operating characteristic of an autoradiographic system determines the relationship between increasing amounts of radioactivity and grain

26 A. W. Rogers, "Techniques of Autoradiography." Elsevier, Amsterdam, 1973. 27 p. Dormer, "Molecular Biology, Biochemistry and Biophysics," p. 347. SpringerVerlag, Berlin and New York, 1973. 28 R. P. Perry, in "Methods in Cell Physiology" (D. M. Prescott, ed.), p. 305. Academic Press, New York, 1969. 29 R. J. Przybylski, in "Introduction to Quantitative Cytochemistry" (G. L. Wied and G. F. Bahr, eds.), p. 477. Academic Press, New York, 1970.

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density (for emulsion autoradiography) or film optical density. 26This need not be linear, but is typically a sigmoid curve whose midsection may approximate a linear function. This operating characteristic provides the most convenient means for approaching data analysis. After film or emulsion hybridization autoradiograms are generated, the amount of film optical density, or grain density, must be related back to the amount of radioactivity. These studies can reveal the nature of the relationship between radioactivity and grain density, on the one hand, and also allow for quantitation of the amount of probe hybridized. This can be performed by coexposing autoradiographic standards of known activity with the unknown samples. Commercially available standards now incorporate tritium and iodine-125 in a medium whose quenching/absorbance parameters resemble those of brain tissue. These standards are available in blocks that can be sectioned at the same thickness as experimental materials and exposed to autoradiographic films or emulsions along with the experimental sections. Alternatively, standards can be constructed by incorporating known amounts of isotope in aliquots of tissue homogenate that can be frozen, cut at the same thickness as experimental samples, and coexposed with unknown samples. 3° In our hands, relatively good ranges of extended nearly linear film and emulsion response to 3H, 125I, or 35S radioisotopes can be obtained. Quantitation corresponding to 32p can yield more strikingly nonlinear responses. Artifacts: Section and Emulsion Thickness Variation Several factors can produce artifactual variation in autoradiographic signals. When using relatively higher energy radioisotopes, such as 35S and 32p, the intensity of the autoradiographic signal will depend not only on the relative cellular and regional concentration of target RNA or DNA, but also on the thickness of the sections and of the emulsion, z6 Thus, tissue areas to which more emulsion adheres can display increased grain numbers based solely on the increased emulsion thickness. For these high-energy isotopes, the amount of radioactivity over a particular area will also be proportional to the section thickness. Thus, attention to section evenness is of substantial importance. One means for minimizing tissue-induced variability in emulsion thickness employs emulsion-coated coverslips. 3~

3o G. Uhl and K. Hill, in "In Situ Hybridization in Brain" (G. R. Uhl, ed.), p. 287. Plenum, New York, 1986. 3~ M. J. Kuhar, in "Neurotransmitter Receptor Binding" (H. I. Yamamura, S. J. Enna, and M. J. Kuhar, eds.), p. 153. Raven, New York, 1985.

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Artifact: Quenching When using tritium, however, a different set of considerations prevails. Different tissue constituents can exert differential "quenching. ''26,32 Differing degrees of tissue self-absorbance of weaker tritium/3 emissions can yield artifactual differences in apparent hybridization densities. Conversely, the relatively low mean free path length of these tritium/3 emissions makes small variations in emulsion and section thickness less crucial, because only the top few micrometers of section thickness and the adjacent few micrometers of emulsion thickness are involved in exposures using this isotope.

Artifacts: Chemography Emulsions can react with nonradioactive chemical tissue constituents. These interactions can produce either artificially high or artificially low grain densities (positive and negative chemography). 26 Tissue sections free from radioactivity or those mounted over a uniform slide coating of isotope can be utilized to check for positive and negative chemography, respectively. Approaches to Quantitation: Analyses

Film Film autoradiograms are best analyzed by using an image analysis system that allows calibrated determination of optical densities over geometrically irregular regions of interest. The borders of these regions can be determined by careful comparison of the film autoradiographic images to cell-stained representations of the tissue section used to generate the autoradiograms. The film optical density will fall off more or less gradually at the edge of an area rich in radioactivity. Thus, standardized conventions should be employed to define the edge of a region, so that the same sampling strategy can be reproduced on each tissue section analyzed. This is of greater concern if the biological system under study can change its cellular characteristics or size. When positively hybridizing cells are scattered over a region, care must be taken to avoid saturation of the small film areas that overlie the cells themselves. Regional film optical densities reflect both the high densities over hybridizing cells and the low densities between the cells. Thus, local film saturation can take place when the optical density values of the 32 W. A. Geary III, A. W. Toga, and G. F. Wooten, Brain Res., in press (1988).

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entire region are substantially below saturation. Determination of hybridization densities over individual cells in emulsion autoradiograms can obviate this potential difficulty. Background optical density values are typically subtracted from each observation. Careful attention to selection of tissue areas known to be free of the mRNA of interest can provide a background determination that takes into account radioactivity that is nonspecifically absorbed or trapped by the tissue. Alternatively, an area of film away from the tissue can provide an estimate of the autoradiographic background that is attributable to the density of the film alone. Optical densities over mRNA-sense and autoradiographic standards coexposed to the same film can also be determined. With these values in hand, the optical density values corresponding to regions of the unknown sample can be related more accurately to the density of radioactivity, the density of hybridized probe, and the density of accessible mRNA.

Emulsion Signal. In studies where the relative cellular content of hybridizable mRNA is of interest, determining the density of autoradiographic grains over an area of a cell can provide a good measure of hybridization density. However, if absolute measurements of total cellular mRNA content are undertaken, then all of the grains associated with the cell must be counted. 26 The distribution of grains from a point source falls off with distance from the source. Thus, the distance from a cell of a grain attributable to a radioactive decay event within the cell will vary with the isotope used. Furthermore, if cells are close together, some of the grains lying over the second cell may be attributable to radioactive events in the first cell, and vice versa. These considerations make absolute determinations of total cellular grains difficult unless (1) the cells are widely separated and (2) the autoradiographic background is so low that virtually all of the grains in the vicinity of the cells can be confidently attributable to specific hybridization. Background. Autoradiographic background values can again be determined over tissue zones free of the mRNA of interest, or from emulsion that overlies no tissue. These grain densities are typically subtracted from the signal observed over positively hybridizing zones. Partial Volume Effects. In studies comparing hybridization to different cell types, it is important that the cells be represented to the same extent in the tissue section. 26 One approach to this problem involves quantitating autoradiographic signals only over cells whose nucleus is

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present in this section." This technique avoids "partial voluming," the inclusion of only the edge of a cell within a small fraction of total section thickness. Data Analysis Regional Values: Film Optical Densities

When the cells of interest lie close together, and when they behave in a homogeneous fashion, the average optical density over the region is the parameter of interest. This value is corrected for background, typically by subtracting the optical density of an adjacent film area that did not lie over a tissue section. Finally, the position of the value on two standard curves is noted. In comparison with the radioactivity standards constructed with the same isotope used to label the hybridization probe, the relation of the value to film saturation and to other radioactivity levels can be assessed. In comparison with cohybridized mRNA-sense standards, the relation between the unknown value and the amount of added mRNA can be determined. If both of these comparisons yield values within a nearly linear range, then the unknown data can be validly compared directly to other background-corrected unknown samples without fear of improper interpretation due to saturation of tile film or hybridization of all available probe molecules. Statements about the absolute density or copy number of mRNA present in the tissue, however, must still be tempered by the realization that tissue factors hindering full hybridization may not be present in the mRNA-sense standards. Statistical treatment of such data is straightforward. Although there is no reason to believe a priori that the data need be parametric, these tests are often used in practice. Cellular Values: Grain Densities from Emulsion Autoradiograms

If quantitation of hybridization to individual neurons is required, the density of autoradiographic grains above the cells of interest is easily determined. This can be done usually using a calibrated eyepiece grid or can be approached using an automated image analysis system. We routinely assess grain counts in a 10 × I0 /xm grid box positioned over neuronal cytoplasm at 100× objective magnification. 33 Alternatively, several image analysis systems allow assessment of grain densities overlying cells of certain size classes. When these figures are corrected for autora33G. R. Uhl and C. A. Sasek, J. Neurosci. 6, 3258 (1986).

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diographic background, they can be displayed as frequency distribution histograms. The fraction of all cells displaying certain grain densities can be plotted against ascending grain density. The numerical data obtained in this way can be analyzed based on such questions as (1) What is the fraction of total cells that express the mRNA of interest, and how does it change with a biological stimulus? and (2) What is the intensity (and range} of expression of the mRNA of interest, and how does this change with various stimuli? Determination of the threshold for considering a cell to be positively expressing can be based on its displaying several times (e.g., 3-5) more grains than background values or on an assessment of the shape of the cell labeling densityfrequency distribution histograms. These numerical values can again be compared directly by simple statistical tests. If one wishes to compare statistically two population hybridization density distributions, more complex statistics such as the Kolmogorov-Smirnov test must be used. These methods are also discussed by McCabe et al. (this volume [59]). Conclusions As approaches to in situ hybridization mature, and as more questions arise about gene regulation in individual cells, quantitative in situ hybridization analyses will assume increasing importance. With careful attention to specific technical details, this approach should yield data that can help to address many of these biological questions. Acknowledgments I thank Drs. Robert Singerand Gerhard Heinrichfor valuablecomments,and Ms. Janice Canifffor assistance with the manuscript. This work was supported by the HowardHughes Medical Institute, McKnightFoundation,SloanFoundation,AmericanParkinson'sDisease Association, and the National Institutes of Health.