In situ hybridization (ISH) plays an important role in the field of molecular diagnostics, especially in an anatomical pathology laboratory. ISH is a technique that can detect the targeted DNA or RNA sequences in tissue sections from frozen or fixed materials with labeled DNA or RNA probes. Radioactive and non-radioactive probes are the two major probes that can be used to label the targeted nucleic acids.

Two decades after the Human Genome Project, ISH has not only simply been applied to identify the chromosomal location of a human gene but has also been extensively applied to gene expressions studies and utilized for clinical diagnosis, especially for the determination of biomarkers for breast and ovarian cancers - human epidermal growth factor receptor 2. Duchenne muscular dystrophy, Cri-du-chat syndrome, Angelman syndrome, PraderWilli syndrome, cystic fibrosis, and trisomy are diseases that can also be detected by ISH. In this review, the basic principles, historical development, advantages and disadvantages, enhancement in reporting molecules and probes, advancement in detection methods, in situ PCR, clinical applications and novel applications of ISH will be discussed.

With the advancement in ISH technologies and appropriate training, diagnosis can be improved in Anatomical Pathology.

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge: E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed: the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool.

Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at -20 degrees C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications.

Kidney-derived aspartic protease-like protein (KAP), initially identified in the mouse kidney, is a novel aspartic protease exclusively expressed in the lung and spleen as well as the kidney. Its orthologues have been identified in the human and rat, and termed napsin. We performed in situ hybridization analysis to determine the cellular expression of napsin mRNA in the kidney, lung, and lymphatic organs of adult mice and to demonstrate, for the first time, its expression patterns in ontogeny. In the adult mouse kidney, extremely intense signals for napsin mRNA were observed in the proximal straight and convoluted tubules, in agreement with a previous study. The first signals for napsin mRNA during nephrogenesis occurred selectively in mesonephric tubules at embryonic day 13, and in metanephric tubules from embryonic day 14. In the lung, a distribution restricted to type II alveolar cells or their precursors was found from embryonic day 15, at the onset of type II cell differentiation, to the adult stage. In the spleen, the mRNA was expressed in lymph nodules of the white pulp and the marginal zone-namely, B-lymphocyte-rich regions from postnatal day 0 to adult. The lymph node and Peyer's patch displayed similar expression patterns, but T cell-dependent areas in these organs and the thymus lacked such signals. These findings suggest that mouse napsin possesses crucial functional roles not only in the kidney but also in the lung and lymphatic tissues, even during fetal stages.

Simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) was achieved by a double-labeling technique using a combination of immunohistochemistry and in situ hybridization in five pigs with naturally occurring porcine dermatitis and nephropathy syndrome (PDNS). Both PRRSV and PCV2 were isolated from a homogenate of pooled skin and kidney from three pigs. PRRSV RNA was demonstrated by reverse transcription polymerase chain reaction (PCR) in skin, kidney, lymph node, and tonsil homogenates from all pigs. PCV2 DNA was demonstrated by PCR in kidney, lymph node, tonsil, liver, and lung homogenates from all pigs. For double-labeling studies, the tissue samples were processed sequentially, first by immunohistochemistry and then by in situ hybridization. The most consistent and intense staining for PRRSV and PCV2 was in the kidney, lymph node, and tonsil. PRRSV antigen and PCV2 DNA were also detected in the skin. This morphologic study is the first to confirm the presence of both PRRSV and PCV2 in the same tissues in pigs with naturally occurring PDNS.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

Epstein-Barr virus (EBV) has been consistently shown to be associated with undifferentiated nasopharyngeal carcinoma (NPC). In this article, the authors attempt to detect Epstein-Barr virus in distant relapsed metastatic sites in undifferentiated NPC patients with cured primary cancer.

In situ hybridization (ISH) technique is a reliable method to detect EBV early RNAs (EBERs) within NPC cells. We used a nonisotopical ISH technique to examine the presence of EBERs in paraffin-embedded tissues obtained from 1 paired specimen of primary NPC and its metastatic counterpart at liver and 2 metastatic specimens of retroperitoneal lymph nodes and bone.

All the primary lesions and the metastatic tumors of NPC with undifferentiated histology contained EBERs that could be clearly detected in the nuclei of cancer cells.

This article shows that EBERs can be successfully detected in cells of the distant relapsed metastatic sites. These results suggest that this nonisotopical ISH method of EBERs can be potentially used to diagnose NPC patients developing distant relapsed metastatic lesions with cured primary cancer early. It can offer quick information as to institute suitable salvage chemotherapy for these patients.

The DNA sequence for Kaposi's sarcoma-associated herpesvirus was originally detected in Kaposi's sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi's sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi's sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt's B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi's sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi's sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi's sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi's sarcoma-associated herpesvirus isolates from primary Kaposi's sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi's sarcoma.

Renin is well-known to be a trigger enzyme in the renin-angiotensin system (RAS). In contrast to the classical RAS, the local RAS has recently been noted in several tissues. The local RAS has a function independent from that of the classical RAS, although its physiological principles are not well known. In the present study, we immunohistochemically demonstrated that hepatocytes in the rat express renin. No renin-immunoreactive cells were detected in rat liver at 0 min after death. At 15 min after death, a small number of renin-positive cells was demonstrated in the lamina hepatica, and they increased with time to the end of observation. Immunoreactivity for renin was scarce throughout the cytoplasm, sometimes condensed to below the cell membrane and around the intracellular granules. Histoplanimetrically, the values from 15 min to 120 min after death were significantly different from that at 0 min after death. Hybridohistochemistry revealed no hybrid signals throughout the liver at either 0 min or 30 min, although renin-immunoreactivity was clearly demonstrated in adjacent sections of liver at 30 min after death. In RT-PCR, the radioactivities in kidney and liver at 0 min after death were not different from those at 30 min after death, respectively. These results suggest the existence of hepatic renin in the rat.

To obtain evidence of renin-synthesizing cells in the murine coagulating gland (CG), CG renin mRNA was detected by hybridohistochemistry, as well as in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) in intact, castrated and testosterone-treated C57BL/6 mice. Hybridohistochemistry using paraffin sections of the kidneys and the CGs for the detection of renin mRNA was performed with digoxigenin-labelled probes. Some paraffin sections were immunohistochemically stained for renin by the peroxidase-anti-peroxidase method. Total RNA was extracted, incubated by reverse transcriptase, and amplified by PCR. In the kidneys, the immunoreactivity and the positive signals of hybridohistochemistry using an antisense probe were restricted to the same juxtaglomerular cells. In the control and at 7 days after testosterone administration to castrated mice, both renin-immunoreactivity and -hybridoreactivity were expressed by the epithelial cells in the CGs, while, in the CGs of the castrated mice and 3 days after testosterone injection of castrated animals, neither renin-immunoreactivity nor -hybridoreactivity was detected in the epithelial cells. Using RT-PCR, renin mRNA from the mice in the control and 7 days after testosterone injection of castrated was amplified, whereas, in the castrated and the 3 days after testosterone injection of castrated groups, it was not detected. The data presented here provide additional evidence that CG renin is regulated by testosterone.

Annexin I is a calcium- and phospholipid-binding protein that is involved in the regulation of cellular differentiation. The aim of the present study was to determine the localization of annexin I mRNA expression in normal and diseased human skin. In situ hybridization with a specific digoxigenin-labelled RNA probe was used throughout. We detected no annexin I mRNA signals in basal and suprabasal cells of normal epidermis, but positive signals were evident in the sudoriferous ducts. Annexin I mRNA expression was detected in the keratinizing squamous cells in keratotic type seborrhoeic keratosis and in keratinocytes at the periphery of the horn pearl in well-differentiated squamous cell carcinoma. Positive signals were also seen at the border between involved and noninvolved skin in psoriasis vulgaris and in dyskeratotic epidermal keratinocytes in keratosis follicularis Darier. By contrast, no annexin I mRNA signals were detected in tumour cells in basal cell carcinoma. The present results suggest that annexin I expression is related to, and may play a role in, keratinization disorders.

Although viremia is an integral part of the pathogenesis of human cytomegalovirus (HCMV) disease, the interaction between HCMV and circulating leukocytes of actively infected patients remains an area of uncertainty. It is still a matter of dispute, whether leukocytes support viral replication with subsequent production of infectious virus. In a new approach we developed and applied a sensitive fluorescence in situ hybridization assay for the precise intracellular localization of HCMV genomes in leukocytes. It was shown that in vivo HCMV genomes were exclusively localized in the cytoplasm of leukocytes, indicating that the majority of these cells are virus carriers or abortively infected. Though this method easily detects single copy genes in metaphase chromosomes, the number of HCMV DNA positive leukocytes was significantly lower than the number of HCMV pp65 antigen positive cells. In relation to the pp65 antigen positive cells, only 1-4% of these cells were DNA positive. In addition, the much lower frequency of HCMV immediate early antigen positive leukocytes in comparison to the pp65 antigen positive cells and the impossibility of detecting other viral antigens support the hypothesis that the origin of pp65 found in leukocytes results mainly from protein uptake.

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.

An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling of in situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in the in situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three- to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling of in situ hybridization products and tissue antigens.

Lymphoepithelioma-like carcinoma, which is an uncommon histological type of epithelial tumor, has been described as being closely associated with Epstein-Barr virus (EBV) infection in organs other than the lung. Recently, we experienced two surgically resected cases of pulmonary tumors mimicking lymphoepithelioma-like carcinoma. Both cases contained EBV DNA genomes as shown by polymerase chain reaction (PCR) using EBV DNA-specific primers, one positive for EBV DNA in virtually all cancer cells, and the other showing positive hybridization in a small number of cancer cells by in situ hybridization (ISH) using digoxigenin-labeled olignucletide probes for each of EBV DNA for EBV DNA. EBV-encoded RNA-1 (EBER-1) was typically detected in one case. These results are highly suggestive of EBV-associated tumors in one of the current cases, although in the other case, no such close association was determined. It seems that lymphoepithelioma-like pulmonary carcinoma, which seems extremely unusual, may be closely associated with EBV infection in tumorigenesis.

Seventeen of 40 cases of Hodgkin's disease (HD) and eight of 46 non-Hodgkin's lymphomas (NHL) were associated with Epstein-Barr virus (EBV) infection, judged by the EBER-1 in situ hybridization (ISH) method. Approximately 40% incidence in HD was comparable to previous reports. Young children and elderly HD patients were more prone to be found EBV positive. Fourteen of 17 HD and two of 8 NHL cases with positive EBER-1 ISH were also positive on LMP-1 immunostaining. EBV might have a role in lymphomagenesis in these cases. The fact that 7 of 8 EBV-related NHL were peripheral T cell lymphoma indicates the necessity of a larger-scale survey on this subject. As the present study revealed four cases with positive LMP-1 immunostaining but negative EBER-1 ISH (1 HD, 3 NHL), LMP-1 alone should not be regarded as a tool to prove EBV infection.

Normal transverse growth of long bones is by periosteal appositional bone formation, balanced by endosteal resorption. Changes in the distribution of cells that are expressing collagen mRNAs during growth were determined using digoxigenin-labelled riboprobes. In neonatal rabbit tibiae osteoblasts expressing type I collagen mRNA are found on periosteal, and at early stages on endosteal, bone surfaces and lining peripheral cavities. Occasional osteocytes express type I collagen mRNA very weakly. The pattern is disrupted when transforming growth factor-beta 2 (TGF-beta 2) is injected daily into the periosteum of neonatal animals; there is increased bone, and later cartilage, formation. Three injections of 20 ng TGF-beta 2 onto the tibia of 3-day-old rabbits led to an increase of periosteal osteoblasts that express the mRNA for type I collagen. Some endosteal osteoblasts and osteocytes in newly-formed peripheral woven bone also express the mRNA. After five injections chondrocytes expressing type II collagen mRNA are found around the injection site. Similar injections of TGF-beta 2 in old rabbits induce only fibrous tissue within which some cells express type I collagen mRNA. This precise localization of mRNAs shows that the expression of type I or II collagen mRNA is here restricted to osteoblasts and chondrocytes, respectively.

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.

Epstein-Barr virus (EBV) has been detected in lymphoepithelioma of nasopharynx and lymphoepitheliomalike carcinomas in various organs. To clarify the association of EBV with gastric carcinoma with lymphoid stroma, which often resembles lymphoepithelioma, the authors examined 22 such cases by polymerase chain reaction and in situ hybridization techniques. In 18 informative cases, EBV DNA was detected by polymerase chain reaction in 14 (77.8%) cases, including lymph node metastases. EBV RNA was detected within the nuclei of carcinoma cells by in situ hybridization in all cases that were positive by polymerase chain reaction. Infiltrating lymphocytes and normal epithelia adjacent to carcinoma were EBV-negative. Southern blot analysis indicated clonal proliferation of tumor cells and episomal form of EBV. These findings suggest that EBV infection occurs before transformation and may be related to oncogenesis of EBV-associated gastric carcinoma.

HPV type 18 DNA was identified in an intrabony ameloblastoma using radiolabelled in situ hybridization. The viral DNA was found in a verrucous lesion in a cystic area of the tumor. The absence of HPV DNA in other epithelial areas of the ameloblastoma is suggestive of a secondary infection. HPV is not considered to be an etiological factor in the pathogenesis of this ameloblastoma.

A case of nasopharyngeal carcinoma is presented. Epstein-Barr viral genome was identified in the neoplastic cells by in situ hybridization with digoxigenin-labelled polymerase chain reaction-generated probes. We report the development of this technique in paraffin-embedded sections and propose that such identification may prove valuable for the diagnosis of this tumour in routine material.

Different approaches to the in-situ polymerase chain reaction (in-situ PCR) were compared in the detection and in-situ localization of chromosomal translocations (t14; 18) immunoglobulin gene rearrangements and viral DNA (cytomegalovirus, hepatitis B-virus) in cell suspensions, cytospins and tissue sections. Single and multiple primer pairs were compared in the amplification step of indirect in-situ PCR and long genomic probes or internal oligonucleotide probes in the subsequent in-situ hybridization (ISH). For direct in-situ PCR, in which amplification products were directly labeled with digoxigenin-11-dUTP during PCR and detected immunohistochemically, only single primer pairs were used for amplification. In-situ PCR yielded best results in the cell suspensions and worked less efficiently in cytospins or tissue sections. Quantification of the results obtained in artificial cell mixtures yielded only an approximate correlation between the number of expected and observed positive cells. The specificity of the results was greater with indirect in-situ PCR than direct in-situ PCR, where false positive results were frequent. Successful indirect in-situ PCR in tissue sections required the use of multiple primer pairs for amplification and genomic probes for detection by ISH. False positive results in direct in-situ PCR were caused by primer-independent, but DNA polymerase- and cycling-dependent incorporation of digoxigenin-labeled nucleotides into cellular DNA, possibly related to DNA repair and/or internal priming. Non-specific results were most marked in tissue sections and were much less frequent in cell suspensions. In-situ PCR includes a number of different techniques, which are not equally applicable to different starting materials. Accurate interpretation of the results requires vigorous controls.

Viral infection has been considered to be a possible pathogenesis of vestibular neuronitis, and reactivation of the herpes simplex virus (HSV) is one of the most likely causes. However, it remains unknown whether the human vestibular ganglia contain latent HSV. We examined 26 vestibular ganglia from autopsied adults in search of HSV type 1 (HSV-1). To detect HSV-1, we used polymerase chain reaction (PCR), in situ hybridization and immunohistochemical staining. HSV DNA was detected in 6 of 10 vestibular ganglia using the PCR method. However, the latency-associated transcript (LAT) of HSV-1 was negative in all of the 16 vestibular ganglia examined. No HSV antigen was detected in any of the ganglia. These results indicate that HSV-1 is latently infected in the human vestibular ganglia, and that LAT is transcribed weakly or not at all.

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti-CD3 (T cells), anti-CD68 (macrophages), anti-CD57 (NK cells), and an anti-pan-B cell antibody (L26). The majority of tumour infiltrating inflammatory cells are T cells (40-50%) and monocytes/macrophages (15-35%). The macrophage specific chemo-attractant and growth factor CSF-1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cells in 46/50 tumours but not at the level of in-situ (pre-invasive) cancer. A mosaic staining pattern was usually observed, with a very high expression in areas of obvious stromal invasion (90% cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cells are equally intensely positive. In-situ hybridisation experiments confirm the production of CSF-1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF-1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF-1 and fms in in-situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF-1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF-1 production by the tumour.

The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

A non-isotopic in situ hybridization procedure was used to detect cytomegalovirus (CMV) sequences within routinely fixed tissue. A digoxigenin-tailed oligonucleotide was hybridized to sections of specimens obtained at autopsy from 2 patients with CMV infection. Hybrids were revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. Serial sections were also assayed for the presence of CMV by in situ hybridization with a biotin-labelled cDNA probe and by immunohistochemistry and routinely stained for morphological evaluation. Results show that the two in situ hybridization procedures are equally sensitive but superior to the immunohistochemical detection of the viral antigen. Most cells positive for CMV DNA had the cytopathological features characteristic of CMV infection. A minor population of infected cells lacking morphological changes was also found. We recommend the routine application of the oligonucleotide-based assay because it is specific, easy and less expensive than other similar procedures.

A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in formalin-fixed, paraffin-embedded liver sections by a nonisotopic in situ hybridization (NISH) procedure. Twenty-three liver samples from chronically HDV-infected patients were studied. Eight liver specimens from humans and chimpanzees without markers of active HDV infection served as negative controls. In three samples, the NISH findings were compared with characteristic nuclear features and with the distribution of the HDV encoded antigen, HDAg, as detected by direct immunofluorescence. All samples from HDV-infected patients were positive for HDV RNA by NISH. The viral genome was exclusively observed within the hepatocytic nuclei. No enzymatic reaction was detected after hybridization with the negative controls. "Sanded" nuclei, a cytopathologic change associated with HDV infection, were HDV RNA-positive, but only a small percentage of infected cells showed that feature. Hepatocytes containing the HDV RNA were sometimes binucleated or exhibited giant nuclei. When HDAg and HDV RNA were sequentially detected within the same sections, the localization of the viral antigen almost completely overlapped with the expression of the HDV transcripts, and vice versa. In conclusion, detection of intrahepatic HDV RNA by NISH is a rapid, sensitive, and specific technique that is easily applicable to routine histopathology and allows correlation of HDV with the morphology of hepatocyte nuclei.

Viral infection, especially by reactivation of herpes simplex virus (HSV) has been considered to be a possible explanation for the pathogenesis of idiopathic peripheral facial nerve palsy (Bell's palsy). We investigated whether the geniculate ganglia of man contain latent HSV type 1 (HSV-1), and compared the frequency of HSV-infected ganglia and that of latently infected neurons in human geniculate ganglia and in trigeminal ganglia. From autopsy specimens of eight adults 15 geniculate ganglia and 16 trigeminal ganglia were examined by means of in situ hybridization and immunohistochemical staining. The HSV-1 genome was detected in 11 of the 15 (71%) geniculate ganglia and in 13 of the 16 (81%) trigeminal ganglia. No HSV antigen was noted in any of the ganglia. The incidence of latently infected neurons was 0.9% in the trigeminal ganglia and 5.3% in the geniculate ganglia. The difference in percentages between the two types of ganglia was significant. Our results suggest that reactivation of latent HSV in the geniculate ganglia is a probable cause of some cases of herpetic stomatitis and of idiopathic peripheral facial nerve palsy.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by polyomavirus JC (JCV). In the majority of cases of PML the cerebrum is mainly affected (cerebral PML) but on rare occasions lesions are restricted to the cerebellum and brain stem (cerebellar PML). We report a rare cerebellar PML case which occurred in a Japanese patient undergoing prolonged hemodialysis treatment. To understand the molecular basis of the viral tissue tropism, we molecularly cloned JCV DNA and compared it with those of cerebral PML. Of ten clones analyzed nine showed identical fragment patterns after digestion with various restriction endonucleases, and we designated these clones Sap-1. It could be shown that the basic structures of the regulatory regions are similar between Sap-1 and isolates from cerebral PML. Restriction endonuclease mapping analysis was used to examine the genetic relationship between Sap-1 and urine-derived isolates containing the archetypal regulatory sequence. We found that Sap-1 was genetically related to an archetypal JCV isolate in Japan.

A rapid technique using a non-radioactive receptor molecule (digoxigenin) for intrahepatic hepatitis B virus (HBV) DNA detection using in situ hybridisation was developed. It can be adapted for use in combination with standard immunohistochemistry for simultaneous detection of both HBV DNA and HBV antigens. The total time required for dual detection of HBV antigens and HBV DNA starting from paraffin wax liver sections was two working days. A good signal to background ratio for the detection of HBV DNA was always obtained using this labelling. This technique is cheap, safe, and relatively simple which makes it an ideal tool for the detection of intrahepatic HBV DNA for both routine diagnostic purposes and in research.

A highly sensitive non-isotopic in situ hybridisation technique was developed for the localisation of Epstein-Barr virus (EBV) in paraffin wax embedded tissue sections. The method uses a repeated sequence of the EBV genome as a probe, labelled with the novel reporter molecule, digoxigenin. The method can identify individual copies of EBV by detection of both EBV DNA and highly localised RNA transcripts. A combination of careful proteolytic digestion of tissue sections, high temperature denaturation of probe and target DNA, and sensitive immunocytochemical detection are used to attain single copy sensitivity. The technique is quicker and simpler to perform than some other methods used for the identification of EBV, and provides simultaneous morphological information which cannot be obtained by methods using tissue extracts. This method permits the investigation of the role of EBV in neoplastic conditions of lymphoid and epithelial cells, and may prove valuable in determining the sites of latent virus in healthy subjects.