Cellular localization of cytomegalovirus nucleic acids in guinea pig salivary glands by in situ hybridization

In situ hybridization with guinea pig cytomegalovirus (CMV) DNA probes was used to detect guinea pig CMV nucleic acid sequences in salivary glands during the course of infection. Optimum conditions for detection of guinea pig CMV gene sequences at the cellular level were determined. The technique of nucleic acid hybridization was compared to antigen detection and histologic examination for cellular localization of guinea pig CMV during acute and chronic infection. Tissue sections fixed for 1 h in 4% paraformaldehyde demonstrated darker specific staining and contained significantly larger numbers of cells positive for guinea pig CMV nucleic acids and antigens than sections fixed longer and in other fixatives. The method of in situ hybridization detected more guinea pig CMV infected cells than did routine histological evaluation. Histologically normal salivary gland duct cells as well as cells bearing typical inclusions were found to contain guinea pig CMV nucleic acids. Guinea pig CMV nucleic acids were also detected although less frequently in cells outside of the ducts. These results suggest that in situ hybridization allows for the detection of histologically inapparent guinea pig CMV infections at the cellular level.     

Co-localization of multiple antigens and specific DNA. A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies.     

Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.     

Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization.

In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesions characteristic of parvovirus infection were assayed for the presence of parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissue in all 10 dogs and in nine of the 10 cats, whereas antigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistochemistry. We conclude that an aetiological diagnosis can be established with a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissue sections.     

Ultrastructural localization of viral nucleic acid by in situ hybridization

In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.     

Effects of lipofuscin on in situ hybridization in human neuronal tissue

In situ hybridization is a highly sensitive technique for detecting nucleic acid sequences within tissues, and is frequently employed in neurovirology. However, this technique requires many appropriate controls in order to recognize and avoid potential artifactual hybridization. We have encountered abundant reaction to lipofuscin in neurons in human peripheral and central nervous systems, using various DNA probes, which could be misinterpreted as positive signals. This pseudohybridization reaction was resistant to treatment with RNase or DNase and was also present in tissue sections treated with hybridization mixture or nuclear autoradiographic emulsion in the absence of any radioactive probes. Characteristics used to distinguish between authentic in situ hybridization and the reaction to neuronal lipofuscin include cellular localization, color, margins and granular appearance, sensitivity to treatment with nucleases and the effect of exposure time on signal intensity. These guidelines should be used to avoid potential misinterpretation of in situ hybridization results with human tissue.     

Ultrastructure of in situ hybridization.

Techniques for the ultrastructural localization of structures identified by in situ hybridization are being developed for both preembedding labeling and labeling on thin sections (postembedding). Successful labeling of both RNA and DNA sequences has been reported in recent years. Biotinylated nucleic acid probes are becoming increasing available. Colloidal gold is the only successful ultrastructural label with meaningful spatial localization, and the best results have been obtained with small (20-5 nm) gold particles. The link between biotinylated nucleic acid probes and gold has been protein A, antibiotin, or avidin binding. The size of the target nucleotide sequence, the size of the probe, and the number of gold particles attached to the labeling protein must be understood before there can be meaningful interpretation of micrographs. In addition, the spatial considerations depend on whether preembedding or postembedding is used.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Use of sulfonated probes for in situ detection of amylase mRNA in formalin-fixed paraffin sections of human pancreas and submaxillary gland

A sulfonated probe and its applicability to in situ hybridization is described and discussed. The DNA probes were modified by introducing an antigenic sulfone group into the cytidine residues of the denatured DNA (Budowsky EI, Sverdlov ED, Monastyrkaya GS: New method of selective and rapid modification of the cytidine residues. FEBS Lett 25:201, 1972). Hybridization of the sulfonated DNA with the target nucleic acid sequences was confirmed by an avidin-biotin peroxidase complex method using a monoclonal antibody specific to sulfonated DNA. The detection limit of this system was estimated to be about 1.25 pg of actual target sequences by dot blot hybridization analysis. When the sulfonated probes of human amylase cDNA were applied to in situ hybridization immunohistochemistry on formalin-fixed paraffin sections of the human pancreas and submaxillary gland, hybridization signals were clearly localized in the cytoplasm of the acinar cells of the pancreas, and in the serous cells of the submaxillary gland. Suitable probe lengths for in situ hybridization immunohistochemistry were between 100 and 800 bases. The in situ hybridization technique utilizing a sulfonated DNA probe is sensitive, simple, and easy to perform and applicable to studies of cell biology.     

Rapid technique of DNA-DNA in situ hybridisation on formalin fixed tissue sections using microwave irradiation.

The relative sensitivities of different protocols for detecting cytomegalovirus nucleic acid sequences in histological specimens, using a biotinylated cDNA probe, were assessed. Several commonly used pre-treatment steps were not essential, nor was the use of a highly sensitive detection system. The choice of enzyme used for proteolytic digestion of tissue seems to be important, and increasing the temperature of denaturation of tissue and probe DNA to above 100 degrees C greatly increased the sensitivity of the method. Difficulties in achieving such high temperatures in a controlled manner were overcome by the use of a rapid microwave heating method that can be used routinely in laboratories. This technique detected cytomegalovirus infections in formalin fixed, paraffin processed tissue sections within a single working day.     

Nonradioactive In Situ Hybridization: Recent Techniques and Applications

In situ hybridization (ISH) has become a standard method for the localization of nucleic adid sequences in chromosomes, single cells, and tissue sections. Nonradioactive ISH has not only elimiated the problems associated with radioactive probes but has also achieved a higher degree of resolution. Advances in probe preparation and labeling, methods have facilitated the general application of ISH. In combination with immunohistochemistry, ISH can provide histological information on gene activity at the DNA, mRNA, and protein levels. Some nonradioactive ISH can simultaneously detect nucleic acid sequences in the same tissue or in a chromosome spread. Advances in ISH technology, including use of the polymerase chain reaction offer both a high sensitivity allowing detection of low levels of gene expression and the cytological localization of gene sequences.     

Ultrastructure of in Situ Hybridization

Techniques for the ultrastructural localization of structures identified by in situ hybridization are being developed for both preembedding labeling and labeling on thin sections (postembedding). Successful labeling of both RNA and DNA sequences has been reported in recent years. Biotinylated nucleic acid probes are becoming increasing available. Colloidal gold is the only successful ultrastructural label with meaningful spatial localization, and the best results have been obtained with small (20–5 nm) gold particles. The link between biotinylated nucleic acid probes and gold has been protein A, antibiotin, or avidin binding. The size of the target nucleotide sequence, the size of the probe, and the number of gold particles attached to the labeling protein must be understood before there can be meaningful interpretation of micrographs. In addition, the spatial considerations depend on whether preembedding or postembedding is used.     

Recent developments in signal amplification methods for in situ hybridization.

In situ hybridization (ISH) allows for the histologic and cytologic localization of DNA and RNA targets. However, the application of ISH techniques can be limited by their inability to detect targets with low copies of DNA and RNA. During the last few years, several strategies have been developed to improve the sensitivity of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after the hybridization is completed. Current approaches involving target amplification (in situ PCR, primed labeling, self-sustained sequence replication), signal amplification (tyramide signal amplification, branched DNA amplification), and probe amplification (padlock probes and rolling circle amplification) are reviewed with emphasis on their applications to bright field microscopy. More recent developments such as molecular beacons and in situ strand displacement amplification continue to increase the sensitivity of in situ hybridization methods. Application of some of these techniques has extended the utility of ISH in diagnostic pathology and in research because of the ability to detect targets with low copy numbers of DNA and RNA.     

A two-colour technique for chromosome in situ hybridization in tissue sections.

By extending non-isotopic in situ hybridization of DNA probes (targeted to metaphase chromosomes or interphase nuclei) to hybridization using tissue sections, additional topological information on the DNA structure and specific alterations can be obtained. We have established a method for the application of two different, chromosome-specific probes labelled with two colour dyes allowing simultaneous detection of two-colour signals. This method was tested in and is applicable to tissue sections of various origins. To demonstrate its sensitivity, prostate carcinomas (either as cryosections or as sections from paraffin blocks) were investigated for the presence or absence of chromosomes 1 and Y. The technique presented here, comparable to immunohistochemical staining, is particularly useful for routine application in diagnostic laboratories and testing of fresh or archival material.     

Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein-Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti-latent membrane protein antibody.

BACKGROUND: The detection of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) may be of diagnostic importance, particularly in cases from nonendemic areas. For cellular localization of viral genomes, cold in situ hybridization methods for the demonstration of EBV-associated NPC remain difficult and relatively insensitive for routinely processed tissues. EXPERIMENTAL DESIGN: The aim of the present study was to assess the importance of tissue processing and the hybridization targets to improve the sensitivity of the cold in situ hybridization method. In situ hybridization was performed in six cases of NPC using three biotinylated EBV cDNA probes (BamHI W/IR1, BamHI Y/EBNA2, XhoI/latent membrane protein) and two cocktails of EBER and BHLF1 oligonucleotides labeled with fluorescein isothiocyanate on routinely fixed and paraffin embedded sections. In two cases, in situ hybridization was also performed on specially processed (ModAMeX) sections. Immunohistochemistry was used to detect EBV-induced antigens using monoclonal antibodies against latent membrane protein, EBNA2 and ZEBRA (BZLF1). RESULTS: All cases showed EBV nucleic acids regardless of the tissue preparation with the three cDNA probes and on routinely processed sections with EBER oligonucleotides. By using cDNA probes, the best EBV DNA signal was obtained with BamHI W without heating of slides in tissue sections processed by ModAMeX, which probably gives rise to large amounts of single stranded DNAs. All cases positive with cDNA probes were found to be positive with EBER oligonucleotides and negative with BHLF1. However, on routinely processed paraffin sections, the signals with EBER oligonucleotides were stronger than with BamHI W cDNA probe. Dual labeling with in situ hybridization and immunohistochemistry showed that the hybridization signals were restricted to malignant epithelial cells. Latent membrane protein expression was detectable in four of six EBV nucleic acid-positive cases on both ModAMeX and routinely processed sections. The anti-EBNA2 and anti-ZEBRA antibodies were found to be negative on the two cases processed by ModAMeX. CONCLUSIONS: Cold in situ hybridization, in particular with EBER oligonucleotides, appears to be more reliable than immunohistochemistry with anti-latent membrane protein antibody to detect EBV in NPC in routine pathology. These findings confirm a distinctive phenotype (latent membrane protein +/-, EBNA2-, ZEBRA-) of EBV-positive NPC. The negative staining for BHLF1 oligonucleotides further supports the viral latency.     

In situ hybridization--application to gene localization and RNA detection

In situ hybridization offers a direct approach for localization and quantitation of nucleic acid sequences in cellular preparations. Recent improvements in technology and methodology make possible the detection of DNA and RNA of relatively low copy number. For example, development of in situ hybridization methods for detection of single copy DNA sequences on mitotic chromosomes has led to general use of this technique for gene mapping of the human genome. More recently, improvements in methodology for detection of low abundancy RNA make possible a facilitated analysis of gene expression, both from cellular genes and exogenous sequences, such as viral genomes. In situ hybridization is now a powerful method for studying nucleic acid organization and function in normal cells, as well as in malignant cells, which should contribute to better understanding of the cell transformation process.     

间接原位PCR检测实验性弓形虫感染病理标本

探讨间接法原位 PCR在弓形虫感染病原学检测中的应用。以 RH株弓形虫速殖子感染小鼠后第 2、4、6天处死 ,取肝脏制备石蜡标本及冰冻切片标本 ,以间接法原位 PCR扩增并检测石蜡标本中的弓形虫 DN A,与原位杂交方法检测冰冻切片标本的结果比较。以间接原位 PCR方法检测 18例标本均得到阳性信号 ,病原体检出率为 10 0 % ,优于原位杂交法 ( 9/18)。间接法原位 PCR为弓形虫病的病理检测提供了一种可行的新方法     

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.     

Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization

A novel method for covalently binding formalin fixed paraffin embedded (FFPE) tissue sections to glass microscope slides is validated suitable for in situ hybridization (ISH). Using the organosilane methodology of Maples (1985), 100% tissue adhesion is reported with no nonspecific probe binding, staining, or autoradiographic artefacts. JC viral nucleic acid sequences are successfully detected in FFPE progressive multifocal leukoencephalopathy brain tissue and the Tm of the hybridized product is estimated. From the Tm the most stringent washing condition resulting in an optimal signal to noise ratio is determined. A comparison is made between currently used methods of tissue adhesion and the proposed organosilane methodology. This methodology greatly facilitates studies of conditions for ISH and elucidation of mechanisms of viral infections requiring consecutive FFPE sections. It is also applicable to studies using cryosections and cultured cells.