Bioconjugate Chem. 1991, 2, 19-25
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Highly Fluorochrome Labeled Gene Probes for Quantitative Tracing of RNA in Individual Cells by in Situ Hybridization Katharina Pachmann,*J Klaus Reinecke,* Bertold Emmerich,? and Eckhard Thielg Medizinische Klinik Innenstadt, University of Munchen, Munchen, Germany, GSF Institute for Immunology, Munchen, Germany, and Klinikum Steglitz, Berlin, Germany. Received June 1, 1990
A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochromelabeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeledgene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.
INTRODUCTION Transcriptional processes play a central part in cell proliferation and differentiation. Quantification of the transcription product, the mRNA, may supply important information concerning maturation of normal and malignant cells. Biochemical methods routinely used for investigation of such processes ( I ) require large and uniform populations for analysis. In addition, by blot hybridization, mRNA content can be determined only from whole populations. In situ hybridization, in contrast, is applicable to relatively small cell populations and can also yield results in heterogeneous populations. Moreover, it allows for identification and location of the RNA of interest in individual cells. Conventional methods (2, 3) using radioactively labeled gene probes are hazardous to handle, time consuming, require special precautions, and do not provide quantitative results. Attempts have been made to develop alternative methods (4-10). Here we present a method employing spacer molecules by which an amplification effect is obtained, allowing attachment of many fluorochromes to a gene probe, in contrast to a method previouslypublished by us (11)which only allowed attachment of one fluorochrome molecule per probe molecule by end labeling. The labeling procedure is rapid and easy and can be traced by fluorescence microscopy immediately and, in addition, offers the possibility of quantifying the results. Quantitation of the RNA of genes coding for receptor molecules, as well as oncogenes, and even viral RNA may thus be performed at the single-cell level. EXPERIMENTAL PROCEDURES
Buffers and Solutions. For fluorochrome coupling a 0.5 M carbonate buffer, adjusted to pH 9.5, was used; other
* Addresscorrespondenceto KatharinaPachmann,Med Klinik Innenstadt, Ziemssenstr 1, D-8000 Munchen 2, Germany. + Medizinische Klinik Innenstadt. 1 GSF Institute for Immunology. 5 Klinikum Steglitz. 1043-1802/91/2902-0019$02.50/0
solutions and buffers used were PBS' containing 0.14 M NaCl and 0.01 M sodium phosphate, pH 7.4; 1 X SSC containing 0.15 M NaCl, 0.015 M sodium citrate, pH 7.5; and T E buffer consisting of 10 mM Tris, 1 mM EDTA, pH 7.4. The hybridization buffer consisted of a mixture of 10mM Tris HC1 (Sigma),pH 7.5,l mM EDTA (Sigma), 600 mM NaC1, 0.02 % Ficoll (Sigma), and 0.02 % polyvinylpyrollidone (Sigma),and according to the experimental conditions, 50 7% deionizedformamide (Merck,Darmstadt) was added or omitted. Labeling of the Spacer Molecule with FITC.2 The principle of the method for labeling is shown in Figure la. Fluorochrome molecules are attached to a spacer and amplifier molecule, a synthetic polypeptide PE13 (Serva, median molecular weight 30 000-40 000), by using a method described by Goldman for conjugating proteins with fluorochromes (12)(see step 1in Figure 1). The fluorochrome FITC was obtained from Fluka (Buchs); 1 mL of polypeptide solution (5 mg/mL) in carbonate buffer was incubated with 7.5-20 kg of FITC dissolved in 1 mL of carbonate buffer for 1 h at room temperature. To remove excess fluorochrome, the conjugated polypeptide was purified over a 0.6 X 25 cm Sephadex G25 (Pharmacia) column which had been equilibrated with PBS. Elution was performed with PBS and the first yellow fraction containing the fluorochrome-labeled PEI was collected. With measurement of the absorbance at 280 nm for the peptide and 490 nm for FITC, the degree of labeling could be deduced from a nomograph (13). Since high labeling of the polypeptide results in nonspecific binding of the labeled complex to cellular components via electrostatic forces (14),the concentration of FITC at which no visible binding of the FITC-PE14 conjugate to cell preparations occurred was determined. We found that if FITC was added to the reaction mixture at concentrations exceeding 2 pg/mg of PEI, leading to a
PBS: phosphate-buffered salt solution. FITC: fluorescein isothiocyanate. PEI: polyethylenimine. 4 FITC-PEI: fluorescein isothiocyanate conjugated to poly1
ethylenimine. 0 1991 American Chemical Society
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Pachmann et al.
Bioconjugate Chem., Vol. 2, No. 1, 1991
a FITC
the fact that both the fraction of unbound polypeptide and the DNA-bound polypeptide were yellow, and the first yellow fraction was collected. The fluorescence intensity of a defined volume of labeled probe solution was then compared to the fluorescence intensity of a known FITC solution microfluorimetrically. Cytological Preparations. Lymphoid cells from peripheral blood of patients with ALL7 were isolated by Ficoll Isopaque separation and washed twice in PBS pH 7.4, and lo6cells sedimented in 10 pL of PBS. The Jurkat and the K562 cell lines were cultured in our laboratory. IgM-producing cells of a mouse hybridoma cell line were FI TC- PEI DNA labelled DNA kindly provided by Dr. U. Kummer (GSF Institut fur Hiimatologie). Cultured cells were harvested and washed twice in PBS. The cell membrane was permeabilized in suspension by adding 500 pL of hypotonic medium (0.9% trisodium citrate) to the sediment. At different times (0-5 min) thereafter the cells were fixed by adding 500 pL of ultrapure acetone. After 2-h fixation the cells were cytocentrifuged directly onto slides a t 520 rpm (8g)at a density allowing a t least three cell diameters of free space between the individual cells or allowed to sediment onto adhesion slides (Bio-Rad). ~ - N - c H - ( c H ~ ),pi-~-B In Situ Hybridization. Hybridization reactions were carried out by modifying a method of Fournier et al. (18). Figure 1. (a) Schematic drawing of the principle for labeling Ten microliters of the reaction mixture was added to each gene probes. Step 1: fluorochrome (FITC)is coupled to the polypeptide (PEI). Step 2: the fluorochrome-labeled polypepslide and sealed under a coverslip with a vulcanizing glue, tide is attached to the DNA probe. (b) Assumed coupling mechand the slides were incubated for 24-96 h in a moist anism with the glutaraldehyde is shown. chamber at 37 "C. The slides were then unsealed in 1 X SSC buffer and washed once in the hybridization buffer binding of more than four molecules FITC/PEI molecule, with 50 % ' formamide for 30 min at 47 "C and once in the this resulted in visually observable nonspecific fluoreshybridization buffer without formamide. They were then cence. washed again in 1 X SSC a t room temperature, covered In a second step the fluorescinated PEI was coupled to with one drop of glycerine, and inspected or measured. the DNA probe (see step 2 in Figure la), modifying a Measurements were performed on a Leitz Orthoplan method described by Renz and Kurz (15) using glutaralmicroscope photometer MPV 2 equipped with a Ploem dehyde as a coupling reagent. The possible mechanism optique for fluorescein and rhodamin illumination. Each of the cross-linking reaction is shown in Figure lb. cell was adjusted into a measuring diaphragm under visual DNA coding for the immunoglobulin p constant fragcont,rol and excited individually for 0.5 s. Fluorescence ment5 (80 base pairs of domain C3 and all of domain C4 intensity was measured through a phototube, and values plus intervening sequences) was kindly provided by Dr. were recorded, transferred to a Commodore CM 8032 and M. Pech (Institutefor Physiological Chemistry, University stored on a floppy disk. Relative intensity values were of Munchen) and produced according to the sequence related to an external fluorescence standard. Fluoropublished by Rabbitts et al. (16). The 920 nucleotide chrome-labeled beads (Becton and Dickinson) were used fragment was used for hybridization. For tracing mRNA as external standard. Background measurements were of the TCR6 a and /3 chains, we used cDNA specific for performed on a cell-free space adjacent to the cells and the constant regions of the TCR chains derived from polysubtracted automatically from the total value. Corrected (A) RNA of the MOLT-3 T cell line (17), Salmon sperm net values were plotted as frequency-distribution diagrams DNA used as negative control probe was obtained from (19). Boehringer (Mannheim), sonicated, and electrophoresed, and fragments were sheared to the same size as the probe, RESULTS cut out, and eluted from the gel. The DNA probe was Determination of Optimal Labeling Conditions. denatured by boiling for 5 min and rapid cooling on ice, The positive signal of a specific DNA probe labeled with and between 1 mg and 10 ng of DNA in 10 pL of the FITC-PEI and the background signal of only the hybridization buffer was added to 10 pL of the FITC-PEI FITC-PEI complex reacted with a cell preparation were and incubated together with 3 pL of glutaraldehyde at compared microfluorimetrically at different F/P8 ratios. different concentrations for 10 min a t 37 "C. Excess The specific signal, as well as the nonspecific signal, binding capacity of the glutaraldehyde was saturated by increased simultaneously over the whole range of the adding 5 pL of 2 9% bovine serum albumin. The probe was applied concentrations, but since the signal-to-background then diluted 1:l (v/v) with hybridization buffer. For ratio remained constant, increasing the concentration of calculation of the amount of fluorochrome molecules bound FITC did not provide a better signal-to-noise ratio. per base pair, the labeled DNA probe was separated over Therefore 2 pg of FITC/mg of PEI, a concentration at a G-100 column from the unbound polypeptide with PBS which no visually observable unspecific binding was as elution buffer. Again, monitoring was facilitated by detected, was usually employed in the subsequent work. PE I
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ALL: acute lymphatic leukemia. F/P ratio: fluorescein to protein ratio.
Hybridization with Fluorochrome-Labeled Probes
Bioconjugate Chem., Vol. 2, No. 1, 1991
21
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Figure 4. Mean values of the fluorescence intensities of 100 cellshybridized witha specific ( 0 )and a controlprobe (0) coupled to different concentrations of PEI-FITC: (a) decrease in fluorescence of both specific and control probe (values + SEM) (b) relation between specific probe and control probe at the different concentrations of PEI-FITC.
The optimal amount of glutaraldehyde for cross-linking the fluorochrome-labeled polypeptide to the gene probe was determined with the hybridoma cell line. The signal of the p-specific probe, as compared to sheared salmon sperm DNA of the same chain length as control probe, was highest a t 0.04% glutaraldehyde (Figure 2). Further increase in glutaraldehyde concentration increased unspecific binding of both the control and the p-specific probe, thus reducing the ratio between the specific and the nonspecific signal. The dependence of the signal on probe concentration was tested in the hybridoma cell line with the FITC-PEI conjugate (5 mg/mL of PEI) at a glutaraldehyde concentration of 0.04 % . Starting from 1pg/lO pL, specific probe and control probe (sheared salmon sperm DNA) were diluted in log 2 steps to the same final concentration. Intensity peaked between 3 and 4 X log 2 dilution (Figure 3) and therefore 100 ng/lO pL was used in the further experiments. At the probe concentration thus determined, the optimal concentration of the PEI-FITC complex for discrimination between specific and control probe was tested. Although there was a decrease in absolute intensity (Figure 4a), the ratio between specific and nonspecific binding increased plateauing over about three dilution steps (Figure 4b). Cell Preparation. Having established the optimal conditions for probe labeling, different cell preparation conditions for hybridization were tested. For accessibility of the RNA, the cells were made porous by hypotonic treatment with trisodium citrate before fixation. Increasing the time of hypotonic treatment lead to an increase
in fluorescence signal, approaching a plateau at 3-4 min. Three minutes of hypotonic treatment were therefore usually chosen before addition of the fixative. Among different fixatives, most of them leading to high autofluorescence, the best results were obtained with acetone. It was added 1:l (v/v) to the cell suspension in hypotonic medium immediately after 3 min of treatment and could be left until use of the cells. Cells can be stored under these conditions for at least 2 weeks in a stoppered vial. They were then either cytocentrifuged or applied to adhesion slides. Determination of Optimal Hybridization Conditions. Two hybridization temperatures, 37 and 45 "C, were assayed. Hybridization was performed with a cocktail with 50% deionized formamide or without formamide in the hybridization buffer. Fluorescence intensity increased continually over time, but cells disintegrated at 45 "C; therefore 37 "C was used in further studies. A microfluorograph of cells reacted with the specific probe is shown in Figure 5. Cell preparations were measured a t different times, and frequency histograms established (Figure 6a). The height of the signal was lower under conditions including formamide, and it may be assumed that only the more specific hybrids are formed; however, the plateau was reached 24 h earlier without formamide (Figure6b). In order to reduce nonspecific hybrid formation, the slides in the subsequent experiments were incubated without formamide but were routinely washed after hybridization in a buffer containing 50% formamide at 47 "C for 30 min. Under these conditions a significant difference between a specific probe ( p ) and a nonspecific probe (salmon sperm
22
Pachmann et al.
Bioconjugafe Chem., Vol. 2, No. 1, 1991
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DNA) could be detected in an ALL after only 6 h of hybridization, but the plateau of the reaction was reached at 48 h (Figure 7). To control the specificityof binding of the fluorochromelabeled probe, digestion of cellular RNA with ribonuclease and hybridization inhibition by preincubation with unlabeled homologous probe were performed (Figure 8). After digestion of the cell preparations of an ALL for 1h at 37 "C with 20 pL of a solution of 10 mg/mL RNase A, the specific signal with the Ta probe was decreased to the level of that of the nonspecific signal. Fluorescence of the ALL cells hybridized with the fluorochrome-labeled Ta probe could be reduced almost to control background levels by preincubation with unlabeled Ta but not with TP probe, with which the signal was comparable to that of the untreated control. Quantification of mRNA. To obtain a quantitative measure of the amount of nucleic acid measurable by our method, the amount of labeled probe which can be discerned from background was determined. In a dilution series, as few as 10 labeled probe molecules included in the measuring diaphragm could clearly by detected, resulting in a fluorescence intensity of 1relative units as compared to the background of 0.2 relative units. In addition, a dilution series of unlabeled denatured probe was prepared, and defined volumes (5 pL) were applied to defined 5-mm areas of adhesion slides which bind cells or molecules via electrostatic forces. These areas were then immersed in 2 % BSA to coat remnant binding sites and subsequently reacted with the fluorochromelabeled probe. They were washed as described for the cells and the fluorescence intensity in the measuring area (10pm diameter) determined. Assuming that the applied
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nucleic acids were quantitatively bound to the test areas, we calculated that between 2 and 20 nucleic acid molecules can be distinguished from the background fluorescence with this method (Figure 9). DISCUSSION
Refined methods have recently been developed to study differentiation and proliferation of normal and malignant cells at the molecular level. They allow us to shed light on the steps that occur during activation at the transcriptional and the translational levels and their presumable deviation during malignant transformation. A t the DNA level, the Southern blot hybridization method (20) lead to the detection of rearrangement of DNA sequences coding for lymphocyte antigen receptors (17,21). Moreover, oncogene rearrangements including
Hybridlzation with Fluorochrome-LabeledProbes
Bioconlugate Chem., Vol. 2, No. 1, 1991 23
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Figure 7. Mean values of common acute leukemia cells hybridized for different times with either the fluorochromelabeled specific cp probe (@) or the control salmon sperm DNA
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Figure 8. Frequency-distribution histograms of cells from a common acute leukemia hybridized with either control probe or a T a probe previously treated with RNase or prehybridized with
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chromosome translocations eventually leading to growth stimulation have been detected by this method (22). Whether or not such genes then become activated can be studied at the mRNA level by Northern blot hybridization ( I ) , but with this method only whole cell populations can be studied and one cannot discriminate between cells of high and low RNA content in heterogeneous populations. This is, however, possible by in situ hybridization (2), which allows analysis of mRNA content at the single-cell level. But, whereas in situ hybridization using radiolabeled gene probes is hampered by difficulties in quan-
Figure 9. Fluorescenceintensitiesmeasured on a dilution series of plasmid DNA of defined concentrationsreacted with the same
fluorochrome-labeledplasmid under the same conditions as for the cells.
tifying the results, labeling with fluorochromes has the advantage of being immediately detectable and quantifyable. With the new fluorochrome-labeling method described here, we can calculate the amount of probe molecules bound per cell from the fluorescence intensity measured fluorimetricallydue to the direct binding of fluorochrome molecules to the probe. A limiting factor for the detection of fluorescence is the amount of fluorochrome molecules attached per probe molecule. Although high labeling will lead to nonspecific binding of the FITC-PEI complex to cellular components via electrostatic forces ( I 4 ) , this does not play a role in microfluorimetry where nonspecific fluorescence can be corrected for. Still in the present report a concentration of FITC leading to no nonspecific direct binding of the complex to the cells was used since increasing the FITCPEI ratio did not further increase the signal to background ratio. The concentration of glutaraldehyde, in contrast, may influence the hybridization properties of the DNA probe by modifying the DNA structure. As shown, this concentration reached an optimum providing the highest binding of a specific versus a nonspecific probe treated in the same way. Also, for probe concentration an optimum was obtained. After the optimum was reached there was no further increase in fluorescence with increasing probe concentration. This indicates that at higher probe concentrations, the PEI-FITC complex was no longer present in excess and less PEI-FITC complex per probe molecule was available. Since the probe-FITC-PEI complex forms a quite large molecule, the porosity of the cell membrane necessary to allow entering of the complex is of importance. In our hands a hypotonic shock as used for chromosome preparation (23) and immediate fixation after 3 min of treatment gave the best results. Prolonged treatment did not lead to increase in fluorescence and resulted in cell loss, obviously due to disruption. The cells were then additionally spread onto slides. Probe size was not a limiting factor for entering the cells and access to the RNA molecules, since we found only a borderline improvement of hybridization with reduced probe length, in contrast to reports by others (24). This may be due to the fact that the longer the strand the more FITC-PEI molecules can be attached, thus improving the detectability of bound molecules. As a fixative, acetone was chosen, because it induced the least autofluorescence. Paraformaldehyde, which was only tested recently, may give comparable results (25).
24
Bioconjugate Chem., Vol. 2, No. 1, 1991
Completion of the hybridization reaction in our hands took 48 h, even if significant differences between control probe and specific probe were already seen after 6 h of incubation. This length of time has been reported as the optimal incubation period (26). Differences in hybridization conditions (temperature and salt concentrations) as well as reannealing of the probe strands and thus exhaustion of the single-stranded probe available for hybridization to cellular nucleic acids may contribute to different extents to the termination of the reaction. Use of unidirectional probes may help to overcome this problem. To avoid further prolongation of hybridization times formamide was omitted from the hybridization mixture, but washing was performed in 50% formamide at 45 "C to reduce low-stringency binding. We can detect down to 10 probe molecules, a number that has been calculated directly from the minimal amount of fluorescent molecules which can be discriminated from autofluorescence of cells. The amount of fluorochromelabeled probe reacting with a slide area was correlated to the amount of homologous probe attached to this area over the range of 4 log dilution steps. Work is in progress to directly control whether the unlabeled nucleic acid strands remain completely bound to the test area in order to establish a standard curve. So far we have not seen any other report describing a comparable quantitative method where an exact discrimination at the individual cell level between background and specific fluorescence is possible. The advantage of the method appears especially in instances where low amounts of RNA are to be detected since any fluorescence intensity exceeding control fluorescence (autofluorescence of the cells plus unspecific binding) is indicative for specific binding. We are able to discriminate at least 10 bound probe molecules from background fluorescence. This number is directly calculated from the fluorescence intensity, in contrast to indirect calculation from northern blots, which may be hampered by the heterogeneous expression of specific mRNA in the investigated population (27) Quantitative in situ hybridization with fluorochromelabeled probes may thus contribute to improvement of our knowledge concerning activation of differentiation genes (28) and oncogenes (29)and in microbiology of the virus load of individual cells (30)and the presented method may increase the sensitivity of the hybridization procedure. ACKNOWLEDGMENT
The skillful technical help of Mrs. Sabine Blieninger is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Projekt Em 20/ 6-2,3) and by the Deutsche Krebshilfe (W25/84/Thl). LITERATURE CITED (1) Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method
for detection of specific RNAs in agarose gels by transfer to diazobenzylmethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354. (2) Gall, J. G., and Pardue, M. L. (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 63, 378-383. (3) Gerhard, D. S., Kawasaki, E. S., Bancroft, F. C., and Szabo, P. (1981) Localization of a unique gene by direct hybridization in situ. Proc. Natl. Acad. Sci. U S A . 78, 3755-3759. (4) Baumann, J. G. J., Wiegant, J., and van Duijn, P. (1983) The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy. J . Histochem. Cytochem. 31, 571-578.
Pachmann et al. (5) Baumann, J. G. J., Wiegant, J., and van Duijn, P. (1981) Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model. J . Histochem. Cytochem. 29,227237. (6) Baumann, J. G. J., Wiegant, J., Borst, P., and van Duijn, P. (1980)A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labeled RNA. E x p . Cell Res. 128, 485-490. (7) Hopman, A. H. N., Wiegand, J., and van Duijn, P. (1986) A new hybrid-cytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of the mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten. Histochem. 84, 169-175. ( 8 ) Landegent, J. E., in de Wal, N. J., van Ommen, G.-J. B., Baas, F., de Vijlder, J. J. M., van Dujin, P., and van der Ploeg, M. (1985) Chromosomal localization of a unique gene bynonautoradiographic in situ hybridization. Nature 317, 175-177. (9) Tchen, P., Fuchs, R. P. P., Sage, E., and Leng, M. (1984) Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc. Natl. Acad. Sci. U.S.A. 81, 3466-3470. (10) van Prooijen-Knegt, A. C., van Hoek, J. F. M., Baumann, J. G. J., van Duijn, P., Wool, I. G., and van der Ploeg, M. (1982) In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure. Exp. Cell Res. 141, 397-407. (11) Pachmann, K. (1987) In situ hybridization with fluorochrome-labeled cloned DNA for quantitative determination of the homologous mRNA in individual cells. J. Mol. Cell Immunol. 3, 13-19. (12) Goldman, M. (1969) In Fluorescent Antibody Methods, p 101 ff, Academic Press, New York and London. (13) Goldman, M. (1969) In Fluorescent Antibody Methods, p 125, Academic Press, New York and London. 14) Brandtzaeg, P. (1975) Rhodamine conjugates: specific and nonspecific binding properties in immunochemistry. In Fifth International Conference on Immunofluorescence and Related Staining Techniques (W. Hijmans, Ed.) Ann. N . Y. Acad. Sci. 245, 35-53. 15) Renz, M., and Kurz, C. (1984) A colorimetric method for DNA hybridization. Nucleic Acids Res. 12, 3435-3444. 16) Rabbitts, T. H., Forster, A., and Milstein, C. P. (1981) Human immunoglobulin heavy chain genes: evolutionary comparisons of cp, c6 and cy genes and associated sequences. Nucleic Acids Res. 18, 4509-4524. (17) Yanagi, Y., Chan, A., Chin, B., Minden, M., and Mak, T. W. (1985) Analysis of cDNA clones specific for human T cells and the a and p chains of the T-cell heterodimer from a human T-cell line. Proc. Natl. Acad. Sci. U.S.A. 82, 3430-3434. (18) Fournier, J. G., Kessous, A., Richer, G., Brechot, C., and Simard, R. (1982)Detection of hepatitis B viral RNAs in human liver tissues by in situ hybridization. Biol. Cell 43, 225-228. (19) Pachmann, K., Dorken, B., Emmerich, B., and Thiel, E. (1988)Quantitation of p mRNA by in situ hydridization reveals a correlation between B-maturation associated antigens and IgM gene activation inacute lymphaticleukemias. Med. Oncol. Tumor Pharmacol. 5, 33-39. (20) Southern, B. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. (21) Hozumi, N., and Tonegawa, S. (1976) Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl. Acad. Sci. U.S.A. 73,36283632. (22) Finger, L. R., Harvey, R. C., Moore, R. C. A., Showe, L. C., and Croce, C. M. (1986)A common mechanism of chromosomal translocation in T- and B-cell neoplasia. Science 234, 982985. (23) Gall, J. G. (1963) Chromosome fibers from an interphase nucleus. Science 139, 120-121. (24) Singer, R. H., and Ward, D. C. (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl. Acad. Sci. U.S.A. 79, 7331-7335.
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(25) Lawrence, J. B., and Singer, R. H. (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acid Res. 13, 1777-1799. (26) Bresser, J., and Evinger-Hodges, M. J. (1987) Comparison and optimization of in situ hybridization procedures yielding rapid sensitive mRNA detections. Gene Anal. Tech. 4 , 89104. (27) Evinger-Hodges, M.-J., Brewer, J., Brouwer, R., Cox, I., Spitzer, G., and Dicke, K. (1988) myc and sis expression in acute myelogenous leukemia. Leukemia 2,45-49. (28) Pachmann, K., Raghavachar, A., Bartram, C., Emmerich,
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B., Thiel, E., and Ziegler-Heitbrock, H. W. L. (1989) T cell receptor alpha expression in B-type chronic lymphatic leukemia. Leukemia 3,497-500. (29) Ascione, R., Sacchi, N., Watson, D. K., Fischer, R. J., Fujiwara, S., Seth, A., and Papas, T. S. (1986) Oncogenes: molecular probes for clinical application in malignant diseases. Gene Anal. Tech. 3, 25-39. (30) Neumann, R., Genersch, E., and Eggers, H. J. (1987) Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus. Virus Res. 7, 93-97. Registry No. Glutaraldehyde; 111-30-8
