Molecular and Cellular Probes (1992) 6, 131-136
Detection of rabies virus mRNA in mouse brain by using in situ hybridization with digoxigenin-labelled RNA probes Alan C . Jackson Departments of Medicine and Microbiology & Immunology, Queen's University, 78 Barrie St ., Kingston, Ontario, Canada K7L 317 (Received 24 July 1991, Accepted 28 August 1991)
A non-isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues . Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used . The method had good sensitivity and low backgrounds, and there was excellent cellular localization of signals . ISH wih digoxigenin-labelled probes was compared with ISH with 3 H-labelled probes . This non-isotopic method of ISH is more convenient than the radiolabelled method, and it is quicker because a long autoradiographic exposure is not required .
KEYWORDS: Encephalitis, in situ hybridization, non-isotopic, nucleic acid hybridization, rabies .
INTRODUCTION detecting rabies virus glycoprotein mRNA in the
Rabies virus antigen can be detected rapidly in infected tissues with immunofluorescence.' -' Immunohistochemical methods, including immunoperoxidase staining, have been developed recently that combine the sensitive detection of viral antigen and preservation of anatomical detail .' Rabies virus
brains of experimentally infected mice with digoxigenin-labelled RNA probes . The sensitivity was similar to ISH with 3H-labelled probes . Non-isotopic methods of ISH will have applications in studies of rabies pathogenesis .
RNA has also been localized in cells and tissues of experimental animals and humans with in situ hybridization (ISH) . e-9 In these studies good cellular localization of ISH signals was obtained with 3 H-labelled RNA probes . ISH with 3 H-labelled probes requires long autoradiographic exposures of days to weeks . Recent
MATERIALS AND METHODS Virus The CVS-11 strain of fixed rabies virus was obtained from Dr William H . Wunner (Wistar Institute, Philadelphia, Pennsylvania) . Stock virus was grown in BHK-
reports of non-isotopic methods of ISH have demonstrated good sensitivity, and these methods are more suitable for wide application because they are technically less difficult, quicker, safer, and less expensive than isotopic methods . 1013 Digoxigenin is a steroid hapten that has been used to label both DNA 14-21 and RNA 20-23 probes for ISH studies . Digoxigenin is a secondary metabolite that occurs exclusively in Digitalis plants, and it has an advantage over other biological materials such as biotin because it does not have an endogenous tissue distribution .24 This report describes a method for 0890-8508/92/020131 +06 $03 .00/0
21 cells, concentrated with a Centriprep-30 ultrafiltration device (Amicon, Danvers, Massachusetts), and contained 1 . 9 x 109 plaque-forming units (pfu) ml -1 .
Animals and inoculations Six-week-old female ICR mice (Charles River Canada Inc., St Constant, Quebec) were used . Mice were
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inoculated intramuscularly in the right masseter muscle with 5 X 10' pfu of CVS in 0 .03 ml of phosphatebuffered saline (PBS) with 2% fetal bovine serum (FBS) . Control mice were inoculated with 0.03 ml of PBS with 2% FBS .
Preparation of tissue sections Mice were sacrificed 4-8 days after inoculation . They were anaesthetized with methoxyflurane and perfused with buffered 4% paraformaldehyde. Brains were removed, immersion fixed in the same fixative for 18 h at 4 ° C, dehydrated, and embedded in paraffin . Coronal sections of brain 6 gm thick were cut on a microtome at multiple levels .
In situ hybridization with digoxigenin-labelled
probes Procedures for RNA labelling and immunologic detection of digoxigenin-labelled RNA were obtained from Boehringer-Mannheim (Mannheim, Germany), and the radiolabelled method of ISH by Jackson & Wunner9 was modified for digoxigenin labelling and detection . A 1 . 5 kb fragment of a cDNA clone containing the coding sequence for the rabies virus glycoprotein (G) of the ERA strain of fixed rabies virus (obtained from Connaught Research Institute, Willowdale, Ontario) 25 was excised with Eco RI and Barn HI and subcloned into the dual promoter-containing pGEM2 vector (Promega, Madison, Wisconsin) as described .' Probes for rabies virus glycoprotein mRNA
10 mm Tris-HCI (pH 7 . 4), 1 mm EDTA, and 0 . 1 % Triton X-100. The mixture was applied to the tissue sections on slides for an overnight incubation at 42 ° C . After hybridization the slides were washed three times in 4 x SSC at room temperature for 5 min, once in 2 x SSC at 4 ° C for 5 min, once in 50% formamide, 0 . 3 M NaCl, 20 mm Tris-HCI (pH 8 . 0), and 2 mm EDTA at 55 ° C for 15 min, once in 2 x SSC at room temperature for 30 min, and once in 0 . 1 x SSC at 55 ° C for 15 min, dehydrated in graded alcohols (each containing 0 . 3 M ammonium acetate), and air dried .
For the immunologic detection, slides were blocked with blocking reagent (Boehringer-Mannheim) at room temperature for 30 min and then incubated for 60 min with a 1 :5000 dilution of polyclonal sheep anti-digoxigenin Fab-fragments conjugated to alkaline phosphatase (BoehringerMannheim) . Washed slides were incubated overnight at room temperature in a solution of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) (Boehringer-Mannheim) . The colour reaction was stopped with 10 mm Tris-HCI (pH 8 .0) and 1 mm EDTA, and the slides were dehydrated and mounted with Permount (Fisher Scientific, Fairlawn, New Jersey) . Controls included : (1) tissue sections pretreated with RNase A (Boehringer-Mannheim) ; (2) ISH on uninfected tissues with the rabies virus RNA probe; and (3) ISH of rabies virus-infected tissues with the RNA probe prepared using the Riboprobe Gemini control template .
In situ hybridization with 'H-labelled probes
were synthesized in the presence of digoxigenin-11UTP (Boehringer-Mannheim) and T7 RNA polymerase (Boehringer-Mannheim) . An irrelevant control template (Riboprobe Gemini positive control template, Promega) was used to prepare digoxigenin-labelled RNA transcripts as a control of the specificity of the hybridization . Deparaffinized slides were pretreated by sequential immersion in 0 . 2N HCI for 20 min, 2 x SSC
ISH was performed on sections adjacent to ones
(1 X SSC =0. 15M NaCl, 0 . 015 M sodium citrate, pH 7 . 4) for 30 min, 10 µg ml - ' proteinase K in 10 mm Tris-HCI, 2 mm CaC1 2 , pH 7 . 4 at 37 ° C for 15 min, 0 . 25% (v/v) acetic anhydride in 0 . 1 M triethanolamine HCI buffer (pH &0) for 10 min, and then were rehydrated in graded alcohols and air dried .
RESULTS
The hybridization mixture contained a 1 :20 dilution of the digoxigenin-labelled RNA transcripts, 50 mm dithiothreitol (DTT), 0 . 3 M NaCl, 50% (v/v) deionized formamide, 10% (w/v) dextran sulphate, 0.2 mg ml - ' sheared salmon sperm DNA, 0 . 125 mg ml - ' tRNA, 0. 02% (w/v) Ficoll, 0 . 02% (w/v) polyvinylpyrrolidone,
examined above (digoxigenin-labelled probes) with 'H-labelled RNA probes for rabies virus glycoprotein mRNA as previously described by Jackson & Wunner. 9 The specific activity of the probe was 6 . 0 x 10' dpm µg - ', and the autoradiographic exposure was for 7 days .
Distribution of rabies virus glycoprotein mRNA ISH with digoxigenin-labelled probes for rabies virus glycoprotein mRNA was very sensitive for the detection of rabies virus-infected cells . Marked infection was demonstrated in the CNS (Fig . 1) . The regional involvement was the same as with the other techniques . The sensitivity was assessed with comparisons of ISH on adjacent sections using 'H-labelled probes (see below) .
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Fig . 1 . ISH was performed with a digoxigenin-labelled probe for rabies virus glycoprotein mRNA on a coronal section of mouse brain 6 days after inoculation with CVS . There is marked staining in the cerebral cortex and in pyramidal neurons of the hippocampus . Magnification x 20 .
ISH signals using digoxigenin-labelled probes had excellent cellular localization . Signal was present in
Fig. 2. ISH for the rabies virus glycoprotein mRNA with a digoxigenin-labelled probe on cerebellar Purkinje cells 4 days after inoculation with CVS . There is staining in the perikarya, but not in the nuclei . Magnification x 1250 .
the perikarya of infected neurons, and not in the nuclei (Figs 2 and 3). Signal was not prominent in either dendrites or axons . However, signal was present in the neuropil in regions adjacent to heavily infected neurons (Figs 3b and 4b), likely due to involvement of neuronal processes . Rabies virus RNA was not observed in non-neuronal cell types and background staining was quite low .
For reasons that are unclear, alkaline hydrolysis of digoxigenin-labelled probes did not increase signals as for 3 H-labelled probes (data not shown) . Hence, alkaline hydrolysis of digoxigenin-labelled probes was not performed . The ISH protocols were not optimized for either time or cost efficiency . The hybridization
Comparison of in situ hybridization with digoxigenin- and 'H-labelled probes
time was longer for the non-isotopic method (overnight vs 4 h), but this was for convenience in scheduling. Preparation of the radiolabelled probe from the linearized template took about 3 h longer than the
Both digoxigenin- and 3 H-labelled probes gave good cellular localization of signal in infected neurons (Figs 2 and 3) . ISH signals were present in the same regions of the CNS with these techniques (Figs 4 and 5) . A similar amount of signal was demonstrated in infected neurons with the digoxigenin-labelled probes, and the number of infected cells identified was similar. Signals were much easier to visualize at low magnification for the digoxigenin-labelled probes than for the 3 H-labelled probes using brightfield optics . However, the signals were strikingly similar when examined under high magnification or when compared at low magnification with 3 H-labelled probes using darkfield optics (Figs 4 and 5) . Backgrounds were quite low with both techniques . The sensitivity of ISH using 3 H-labelled and digoxigenin-labelled probes was quite similar. The protocols of both methods were developed for high specificity, which was demonstrated with the controls .
non-isotopic labelling. When the two methods were performed in runs of 30 slides, the technical time for the post-hybridization steps was 4-5 h longer (10 vs 5 . 5 h) for the radiolabelled method . The main difference was that the autoradiographic exposure for the 3 H-labelled probes required a week, and the colour reaction for the non-isotopic method used only an overnight incubation . Hence, the radiolabelled technique of ISH with 3 H-probes was much more time consuming than the non-isotopic technique . The cost of performing ISH, estimated for 1000 slides in runs of 30 slides, was over two times greater for the radiolabelled method .
DISCUSSION Isotopic methods of ISH for the detection of rabies virus genomic RNA and mRNAs for the five rabies virus proteins were reported previously ." A non-
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A. C. Jackson
(a)
(a)
(b)
(b)
Fig. 3. ISH for rabies virus glycoprotein mRNA with a 'H-labelled probe (a) and with a digoxigenin-labelled probe (b) in the brainstem tegmentum 4 days after inoculation with CVS . Three large neurons show marked signals in the perikarya using both methods of ISH, although the amount of signal is greater for the digoxigenin-labelled probe (b) than the 3 H-labelled probe (a) . There is some signal in the neuropil with the digoxigenin-labelled probe (b), which is likely in infected neuronal processes. Magnification x 900 ; a, haematoxylin .
Fig. 4. ISH for rabies virus glycoprotein mRNA with a 'H-labelled probe with darkfield optics (a) and a digoxigenin-labelled probe (b) in deep cerebellar nuclei 4 days after inoculation with CVS . Signals are present in a similar number of infected neurons . The 3 H-labelled (a) and digoxigenin-labelled (b) probes demonstrate similar ISH signals, although the signal is much better visualized at this magnification with brightfield optics for the digoxigenin-labelled probe . Signal is present in the neuropil using the digoxigenin-labelled probe (b). Magnification X 90 .
isotopic method of ISH was developed for the detection of rabies virus mRNA, because non-isotopic methods are more suitable for wide application .
Rabies virus mRNA was localized with good sensitivity by using ISH with digoxigenin-labelled probes . The distribution of signals in the CNS was similar to that found with 3 H-labelled probes . Cellular localiza-
Digoxigenin-labelled RNA probes were convenient because RNA probes were used for the isotopic method, and RNA probes have certain advantages over DNA probes . 8 - 16,11 Since rabies virus is a negative strand RNA virus, either genomic RNA or mRNA can be detected selectively with single-stranded RNA probes . Backgrounds are also low with RNA probes, and digoxigenin lacks an endogenous distribution in tissues .
tion of signals was excellent, and background staining was minimal . The alkaline phosphatase-catalysed colour reaction produces a purple/blue precipitate identifying the signal . The slides were not counterstained with haematoxylin, because haematoxylin is also blue. If it is necessary, then alternative counterstains could be used ." The comparisons of signals with ISH using 3 H-
Detection of rabies virus mRNA
135
In summary, ISH with digoxigenin-labelled RNA probes is a sensitive and specific technique for detecting rabies virus mRNA in tissues . The diagnostic sensitivity was not compared with immunofluorescence, which is even quicker and much more suitable for rapid diagnosis . This non-isotopic method of ISH will have applications in studies of rabies pathogenesis, and also could be adapted for the detection of other viral nucleic acids or cellular mRNAs in cells or tissues .
ACKNOWLEDGEMENTS
(a)
We thank Connaught Research Institute for the cDNA clone containing the coding sequence for the rabies virus glycoprotein and Boehringer-Mannheim for the digoxigenin RNA labelling and detection kits . The technical assistance of Linda Tremblay and secretarial assistance of Martha Steacy are gratefully acknowledged . This work was supported by grant MA-10068 from the Medical Research Council of Canada and the Violet E . Powell Fund (Queen's University) .
REFERENCES
(b) Fig. 5. ISH for rabies virus glycoprotein mRNA with a 3 H-labelled probe using darkfield optics (a) and a digoxigenin-labelled probe (b) in cerebral cortex 6 days after inoculation with CVS . The two techniques demonstrate a similar extent of infection . Magnification X 70 .
labelled probes were favourable for the digoxigeninlabelled probes . The main advantages of the nonisotopic method of ISH are simplicity, speed, safety, and cost . An inability to quantify the signals is a disadvantage of the non-isotopic method if this is important . Double labelling of rabies virus-infected cells can be performed by combining isotopic ISH and immunohistochemical staining as reported previously .' Double labelling procedures have been developed combining digoxigenin-labelled DNA probes and either radiolabelled probes" or biotin-labelled probes ." This suggests that there will be great flexibility for co-localizing specific RNA species or proteins within individual cells .
1 . Kissling, R . E. (1975) . The fluorescent antibody test in rabies . In The Natural History of Rabies . (Baer, C . M ., ed .) pp . 401-16 . New York : Academic Press . 2. Trimarchi, C . V . & Debbie, J . G . (1991) . The fluorescent antibody in rabies . In The Natural History of Rabies . (Baer, C. M ., ed .) 2nd edn, pp. 219-33 . Boca Raton, Florida : CRC Press . 3 . Dean, D . J . & Abelseth, M . K . (1973) . The fluorescent antibody test. In Laboratory Techniques in Rabies . (Kaplan, M . M . & Koprowski, H ., eds) 3rd edn, pp . 73-84 . Geneva : World Health Organization . 4. Feiden, W ., Feiden, U., Gerhard, L ., Reinhardt, V . & Wandeler, A . (1985). Rabies encephalitis : Immunohistochemical investigations . Clinical Neuropathology 4, 156-64 . 5. Fekadu, M., Greer, P . W ., Chandler, F . W . & Sanderlin, D . W . (1988) . Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues . Journal of Virological Methods 19,91-6 . 6 . Feiden, W ., Kaiser, E ., Gerhard, L . et al . (1988). Immunohistochemical staining of rabies virus antigen with monoclonal and polyclonal antibodies in paraffin tissue sections . Zentralblatt fur Veterinarmedizin Reihe B 35, 247-55 . 7 . Jackson, A . C . (1991) . Biological basis of rabies virus neurovirulence in mice : Comparative pathogenesis study using the immunoperoxidase technique . Journal of Virology 65, 537-40. 8 . Jackson, A . C ., Reimer, D . L . & Wunner, W . H . (1989) . Detection of rabies virus RNA in the central nervous system of experimentally infected mice using in situ hybridization with RNA probes . Journal of Virological Methods 25, 1-11 .
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9 . Jackson, A . C . & Wunner, W. H . (1991). Detection of rabies virus genomic RNA and mRNA in mouse and human brains by using in situ hybridization . journal of Virology 65, 2839-44 . 10 . Allan, G . M., Todd, D., Smyth, J . A ., Mackie, D . P ., Burns, J . & McNulty, M . S . (1989) . In situ hybridization : an optimised detection protocol for a biotinylated DNA probe renders it more sensitive than a comparable 35 Slabelled probe . journal of Virological Methods 24, 18190 . 11 . Seyda, M ., Scheele, T., Neumann, R . & Krueger, C . R . F. (1989) . Comparative evaluation of non-radioactive in situ hybridization techniques for pathologic diagnosis of viral infection . Pathology, Research and Practice 184, 18-26. 12 . Syrjanen, S ., Partanen, P ., Mantyjarvi, R . & Syrjanen, K . (1988) . Sensitivity of in situ hybridization techniques using biotin- and 35 S-labelled human papillomavirus (HPV) DNA probes . Journal of Virological Methods 19, 225-38 . 13 . Unger, E . R., Hammer, M . L. & Chenggis, M . L. (1991) . Comparison of 35 S and biotin as labels for in situ hybridization : use of an HPV model system . Journal of Histochemistry and Cytochemistry 39, 145-50 . 14 . Samoszuk, M. & Nansen, L . (1990) . Detection of interleukin-5 messenger RNA in Reed-Sternberg cells of Hodgkin's disease with eosinophilia . Blood 75, 13-16 . 15 . Herrington, C . S ., Burns,] ., Graham, A . K ., Bhatt, B . & McGee, J . O'D . (1989). Interphase cytogenetics using biotin and digoxigenin labelled probes II : Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei . journal of Clinical Pathology 42, 601-6 . 16 . Herrington, C . S ., Burns, J ., Graham, A . K ., Evans, M . & McGee, J . O'D . (1989) . Interphase cytogenetics using biotin and digoxigenin labelled probes I : Relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells . journal of Clinical Pathology 42, 592600 . 17 . Lewis, M . E ., Robbins, E., Grega, D . & Baldino, F . (1990). Nonradioactive detection of vasopressin and somatostatin mRNA with digoxigenin-labelled oligonucleotide probes. Annals of the New York Academy of Sciences 579,246-53 . 18 . Young, W . S . (1989) . Simultaneous use of digoxigeninand radiolabelled oligodeoxyribonucleotide probes for hybridization histochemistry . Neuropeptides 13, 271-5 .
19 . Baldino, F . & Lewis, M. E . (1989) . Nonradioactive in situ hybridization histochemistry with digoxigenin-deoxyuridine 5'-triphosphate-labelled oligonucleotides . In Methods in Neuroscience . (Conn, P. M.,ed.) Vol . 1, pp . 282-92 . New York : Academic Press, Inc . 20 . Hukkanen, V., Heino, P ., Sears, A. E . & Roizman, B. (1990) . Detection of herpes simplex virus latencyassociated RNA in mouse trigeminal ganglia by in situ digoxigeninhybridization using nonradioactive labelled DNA and RNA probes . Methods in Molecular and Cellular Biology 2, 70-81 . 21 . Bochenek, B. & Hirsch, A . M . (1990) . In-situ hybridization of nodulin mRNAs in root nodules using nonradioactive probes . Plant Molecular Biology Report 8, 237-48 . 22 . Hemmati-Brivanlou, A ., Frank, D ., Bolce, M. E ., Brown, B . D ., Sive, H . L . & Harland, R . M . (1990) . Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325-30 . 23 . Springer, J . E ., Robbins, E ., Gwag, B . J., Lewis, M . E . & Baldino, F . (1991). Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry . Journal of Histochemistry and Cytochemistry 39, 231-4. 24. Kessler, C. (1991) . The digoxigenin :anti-digoxigenin (DIG) technology : a survey on the concept and realization of a novel bioanalytical indicator system . Molecular and Cellular Probes 5, 161-205 . 25 . Malek, L . T ., Soostmeyer, G ., Garvin, R. T . & James, E . (1984) . The rabies glycoprotein gene is expressed in Escherichia coli as a denatured polypeptide. In Modern Approaches to Vaccines : Molecular and Chemical Basis of Virus Virulence and Immunogenicity . (Chanock, R . M . & Lerner, R . A., eds) pp . 203-8. New York: Cold Spring Harbor Laboratory . 26 . Moench, T . R . (1987). In situ hybridization . Molecular and Cellular Probes 1, 195-205. 27 . Angerer, L. M ., Stoler, M . H ., & Angerer, R . C . (1987) . In situ hybridization with RNA probes : an annotated recipe . In In Situ Hybridization: Applications to Neurobiology . (Velentino, K . L ., Eberwine, J . H . & Barchas, J . D ., eds) pp . 42-70 . New York : Oxford University Press . 28. Drury, R . A . B . & Wallington, E . A . (eds) (1967) . General staining procedures . In Carleton's Histological Technique . 4th edn, pp . 114-37 . New York : Oxford University Press .
Detection of rabies virus mRNA in mouse brain by using in situ hybridization with digoxigenin-labelled RNA probes Alan C . Jackson Departments of Medicine and Microbiology & Immunology, Queen's University, 78 Barrie St ., Kingston, Ontario, Canada K7L 317 (Received 24 July 1991, Accepted 28 August 1991)
A non-isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues . Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used . The method had good sensitivity and low backgrounds, and there was excellent cellular localization of signals . ISH wih digoxigenin-labelled probes was compared with ISH with 3 H-labelled probes . This non-isotopic method of ISH is more convenient than the radiolabelled method, and it is quicker because a long autoradiographic exposure is not required .
KEYWORDS: Encephalitis, in situ hybridization, non-isotopic, nucleic acid hybridization, rabies .
INTRODUCTION detecting rabies virus glycoprotein mRNA in the
Rabies virus antigen can be detected rapidly in infected tissues with immunofluorescence.' -' Immunohistochemical methods, including immunoperoxidase staining, have been developed recently that combine the sensitive detection of viral antigen and preservation of anatomical detail .' Rabies virus
brains of experimentally infected mice with digoxigenin-labelled RNA probes . The sensitivity was similar to ISH with 3H-labelled probes . Non-isotopic methods of ISH will have applications in studies of rabies pathogenesis .
RNA has also been localized in cells and tissues of experimental animals and humans with in situ hybridization (ISH) . e-9 In these studies good cellular localization of ISH signals was obtained with 3 H-labelled RNA probes . ISH with 3 H-labelled probes requires long autoradiographic exposures of days to weeks . Recent
MATERIALS AND METHODS Virus The CVS-11 strain of fixed rabies virus was obtained from Dr William H . Wunner (Wistar Institute, Philadelphia, Pennsylvania) . Stock virus was grown in BHK-
reports of non-isotopic methods of ISH have demonstrated good sensitivity, and these methods are more suitable for wide application because they are technically less difficult, quicker, safer, and less expensive than isotopic methods . 1013 Digoxigenin is a steroid hapten that has been used to label both DNA 14-21 and RNA 20-23 probes for ISH studies . Digoxigenin is a secondary metabolite that occurs exclusively in Digitalis plants, and it has an advantage over other biological materials such as biotin because it does not have an endogenous tissue distribution .24 This report describes a method for 0890-8508/92/020131 +06 $03 .00/0
21 cells, concentrated with a Centriprep-30 ultrafiltration device (Amicon, Danvers, Massachusetts), and contained 1 . 9 x 109 plaque-forming units (pfu) ml -1 .
Animals and inoculations Six-week-old female ICR mice (Charles River Canada Inc., St Constant, Quebec) were used . Mice were
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inoculated intramuscularly in the right masseter muscle with 5 X 10' pfu of CVS in 0 .03 ml of phosphatebuffered saline (PBS) with 2% fetal bovine serum (FBS) . Control mice were inoculated with 0.03 ml of PBS with 2% FBS .
Preparation of tissue sections Mice were sacrificed 4-8 days after inoculation . They were anaesthetized with methoxyflurane and perfused with buffered 4% paraformaldehyde. Brains were removed, immersion fixed in the same fixative for 18 h at 4 ° C, dehydrated, and embedded in paraffin . Coronal sections of brain 6 gm thick were cut on a microtome at multiple levels .
In situ hybridization with digoxigenin-labelled
probes Procedures for RNA labelling and immunologic detection of digoxigenin-labelled RNA were obtained from Boehringer-Mannheim (Mannheim, Germany), and the radiolabelled method of ISH by Jackson & Wunner9 was modified for digoxigenin labelling and detection . A 1 . 5 kb fragment of a cDNA clone containing the coding sequence for the rabies virus glycoprotein (G) of the ERA strain of fixed rabies virus (obtained from Connaught Research Institute, Willowdale, Ontario) 25 was excised with Eco RI and Barn HI and subcloned into the dual promoter-containing pGEM2 vector (Promega, Madison, Wisconsin) as described .' Probes for rabies virus glycoprotein mRNA
10 mm Tris-HCI (pH 7 . 4), 1 mm EDTA, and 0 . 1 % Triton X-100. The mixture was applied to the tissue sections on slides for an overnight incubation at 42 ° C . After hybridization the slides were washed three times in 4 x SSC at room temperature for 5 min, once in 2 x SSC at 4 ° C for 5 min, once in 50% formamide, 0 . 3 M NaCl, 20 mm Tris-HCI (pH 8 . 0), and 2 mm EDTA at 55 ° C for 15 min, once in 2 x SSC at room temperature for 30 min, and once in 0 . 1 x SSC at 55 ° C for 15 min, dehydrated in graded alcohols (each containing 0 . 3 M ammonium acetate), and air dried .
For the immunologic detection, slides were blocked with blocking reagent (Boehringer-Mannheim) at room temperature for 30 min and then incubated for 60 min with a 1 :5000 dilution of polyclonal sheep anti-digoxigenin Fab-fragments conjugated to alkaline phosphatase (BoehringerMannheim) . Washed slides were incubated overnight at room temperature in a solution of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) (Boehringer-Mannheim) . The colour reaction was stopped with 10 mm Tris-HCI (pH 8 .0) and 1 mm EDTA, and the slides were dehydrated and mounted with Permount (Fisher Scientific, Fairlawn, New Jersey) . Controls included : (1) tissue sections pretreated with RNase A (Boehringer-Mannheim) ; (2) ISH on uninfected tissues with the rabies virus RNA probe; and (3) ISH of rabies virus-infected tissues with the RNA probe prepared using the Riboprobe Gemini control template .
In situ hybridization with 'H-labelled probes
were synthesized in the presence of digoxigenin-11UTP (Boehringer-Mannheim) and T7 RNA polymerase (Boehringer-Mannheim) . An irrelevant control template (Riboprobe Gemini positive control template, Promega) was used to prepare digoxigenin-labelled RNA transcripts as a control of the specificity of the hybridization . Deparaffinized slides were pretreated by sequential immersion in 0 . 2N HCI for 20 min, 2 x SSC
ISH was performed on sections adjacent to ones
(1 X SSC =0. 15M NaCl, 0 . 015 M sodium citrate, pH 7 . 4) for 30 min, 10 µg ml - ' proteinase K in 10 mm Tris-HCI, 2 mm CaC1 2 , pH 7 . 4 at 37 ° C for 15 min, 0 . 25% (v/v) acetic anhydride in 0 . 1 M triethanolamine HCI buffer (pH &0) for 10 min, and then were rehydrated in graded alcohols and air dried .
RESULTS
The hybridization mixture contained a 1 :20 dilution of the digoxigenin-labelled RNA transcripts, 50 mm dithiothreitol (DTT), 0 . 3 M NaCl, 50% (v/v) deionized formamide, 10% (w/v) dextran sulphate, 0.2 mg ml - ' sheared salmon sperm DNA, 0 . 125 mg ml - ' tRNA, 0. 02% (w/v) Ficoll, 0 . 02% (w/v) polyvinylpyrrolidone,
examined above (digoxigenin-labelled probes) with 'H-labelled RNA probes for rabies virus glycoprotein mRNA as previously described by Jackson & Wunner. 9 The specific activity of the probe was 6 . 0 x 10' dpm µg - ', and the autoradiographic exposure was for 7 days .
Distribution of rabies virus glycoprotein mRNA ISH with digoxigenin-labelled probes for rabies virus glycoprotein mRNA was very sensitive for the detection of rabies virus-infected cells . Marked infection was demonstrated in the CNS (Fig . 1) . The regional involvement was the same as with the other techniques . The sensitivity was assessed with comparisons of ISH on adjacent sections using 'H-labelled probes (see below) .
Detection of rabies virus mRNA
133
Fig . 1 . ISH was performed with a digoxigenin-labelled probe for rabies virus glycoprotein mRNA on a coronal section of mouse brain 6 days after inoculation with CVS . There is marked staining in the cerebral cortex and in pyramidal neurons of the hippocampus . Magnification x 20 .
ISH signals using digoxigenin-labelled probes had excellent cellular localization . Signal was present in
Fig. 2. ISH for the rabies virus glycoprotein mRNA with a digoxigenin-labelled probe on cerebellar Purkinje cells 4 days after inoculation with CVS . There is staining in the perikarya, but not in the nuclei . Magnification x 1250 .
the perikarya of infected neurons, and not in the nuclei (Figs 2 and 3). Signal was not prominent in either dendrites or axons . However, signal was present in the neuropil in regions adjacent to heavily infected neurons (Figs 3b and 4b), likely due to involvement of neuronal processes . Rabies virus RNA was not observed in non-neuronal cell types and background staining was quite low .
For reasons that are unclear, alkaline hydrolysis of digoxigenin-labelled probes did not increase signals as for 3 H-labelled probes (data not shown) . Hence, alkaline hydrolysis of digoxigenin-labelled probes was not performed . The ISH protocols were not optimized for either time or cost efficiency . The hybridization
Comparison of in situ hybridization with digoxigenin- and 'H-labelled probes
time was longer for the non-isotopic method (overnight vs 4 h), but this was for convenience in scheduling. Preparation of the radiolabelled probe from the linearized template took about 3 h longer than the
Both digoxigenin- and 3 H-labelled probes gave good cellular localization of signal in infected neurons (Figs 2 and 3) . ISH signals were present in the same regions of the CNS with these techniques (Figs 4 and 5) . A similar amount of signal was demonstrated in infected neurons with the digoxigenin-labelled probes, and the number of infected cells identified was similar. Signals were much easier to visualize at low magnification for the digoxigenin-labelled probes than for the 3 H-labelled probes using brightfield optics . However, the signals were strikingly similar when examined under high magnification or when compared at low magnification with 3 H-labelled probes using darkfield optics (Figs 4 and 5) . Backgrounds were quite low with both techniques . The sensitivity of ISH using 3 H-labelled and digoxigenin-labelled probes was quite similar. The protocols of both methods were developed for high specificity, which was demonstrated with the controls .
non-isotopic labelling. When the two methods were performed in runs of 30 slides, the technical time for the post-hybridization steps was 4-5 h longer (10 vs 5 . 5 h) for the radiolabelled method . The main difference was that the autoradiographic exposure for the 3 H-labelled probes required a week, and the colour reaction for the non-isotopic method used only an overnight incubation . Hence, the radiolabelled technique of ISH with 3 H-probes was much more time consuming than the non-isotopic technique . The cost of performing ISH, estimated for 1000 slides in runs of 30 slides, was over two times greater for the radiolabelled method .
DISCUSSION Isotopic methods of ISH for the detection of rabies virus genomic RNA and mRNAs for the five rabies virus proteins were reported previously ." A non-
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A. C. Jackson
(a)
(a)
(b)
(b)
Fig. 3. ISH for rabies virus glycoprotein mRNA with a 'H-labelled probe (a) and with a digoxigenin-labelled probe (b) in the brainstem tegmentum 4 days after inoculation with CVS . Three large neurons show marked signals in the perikarya using both methods of ISH, although the amount of signal is greater for the digoxigenin-labelled probe (b) than the 3 H-labelled probe (a) . There is some signal in the neuropil with the digoxigenin-labelled probe (b), which is likely in infected neuronal processes. Magnification x 900 ; a, haematoxylin .
Fig. 4. ISH for rabies virus glycoprotein mRNA with a 'H-labelled probe with darkfield optics (a) and a digoxigenin-labelled probe (b) in deep cerebellar nuclei 4 days after inoculation with CVS . Signals are present in a similar number of infected neurons . The 3 H-labelled (a) and digoxigenin-labelled (b) probes demonstrate similar ISH signals, although the signal is much better visualized at this magnification with brightfield optics for the digoxigenin-labelled probe . Signal is present in the neuropil using the digoxigenin-labelled probe (b). Magnification X 90 .
isotopic method of ISH was developed for the detection of rabies virus mRNA, because non-isotopic methods are more suitable for wide application .
Rabies virus mRNA was localized with good sensitivity by using ISH with digoxigenin-labelled probes . The distribution of signals in the CNS was similar to that found with 3 H-labelled probes . Cellular localiza-
Digoxigenin-labelled RNA probes were convenient because RNA probes were used for the isotopic method, and RNA probes have certain advantages over DNA probes . 8 - 16,11 Since rabies virus is a negative strand RNA virus, either genomic RNA or mRNA can be detected selectively with single-stranded RNA probes . Backgrounds are also low with RNA probes, and digoxigenin lacks an endogenous distribution in tissues .
tion of signals was excellent, and background staining was minimal . The alkaline phosphatase-catalysed colour reaction produces a purple/blue precipitate identifying the signal . The slides were not counterstained with haematoxylin, because haematoxylin is also blue. If it is necessary, then alternative counterstains could be used ." The comparisons of signals with ISH using 3 H-
Detection of rabies virus mRNA
135
In summary, ISH with digoxigenin-labelled RNA probes is a sensitive and specific technique for detecting rabies virus mRNA in tissues . The diagnostic sensitivity was not compared with immunofluorescence, which is even quicker and much more suitable for rapid diagnosis . This non-isotopic method of ISH will have applications in studies of rabies pathogenesis, and also could be adapted for the detection of other viral nucleic acids or cellular mRNAs in cells or tissues .
ACKNOWLEDGEMENTS
(a)
We thank Connaught Research Institute for the cDNA clone containing the coding sequence for the rabies virus glycoprotein and Boehringer-Mannheim for the digoxigenin RNA labelling and detection kits . The technical assistance of Linda Tremblay and secretarial assistance of Martha Steacy are gratefully acknowledged . This work was supported by grant MA-10068 from the Medical Research Council of Canada and the Violet E . Powell Fund (Queen's University) .
REFERENCES
(b) Fig. 5. ISH for rabies virus glycoprotein mRNA with a 3 H-labelled probe using darkfield optics (a) and a digoxigenin-labelled probe (b) in cerebral cortex 6 days after inoculation with CVS . The two techniques demonstrate a similar extent of infection . Magnification X 70 .
labelled probes were favourable for the digoxigeninlabelled probes . The main advantages of the nonisotopic method of ISH are simplicity, speed, safety, and cost . An inability to quantify the signals is a disadvantage of the non-isotopic method if this is important . Double labelling of rabies virus-infected cells can be performed by combining isotopic ISH and immunohistochemical staining as reported previously .' Double labelling procedures have been developed combining digoxigenin-labelled DNA probes and either radiolabelled probes" or biotin-labelled probes ." This suggests that there will be great flexibility for co-localizing specific RNA species or proteins within individual cells .
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