In Situ Hybridization Technique Research Articles

Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization.

In order to improve the identification and characterization of dopaminoceptive neurons, the rat brain was mapped for D1 dopamine receptor mRNA by non-radioactive in situ hybridization (ISH) with a 45mer digoxigenin-labeled oligonucleotide probe. The specificity of the results was controlled with the help of a 396-bp D1 receptor riboprobe. Labeled hybrids were visualized with an alkaline phosphatase-coupled anti-digoxigenin antibody. The high resolution obtained permitted individual labeled cells to be identified and to distinction between cell bodies and processes. D1 mRNA was largely confined to neurons. With the exception of ependymal cells, glial cells were not distinctly labeled. Subcellularly, D1 mRNA was localized to perikarya but not to dendrites or axons. D1 receptor-expressing neurons were present in all of the known terminal fields of mesencephalic or diencephalic dopaminergic neurons. However, D1 message was also detected in brain areas which are not known to contain D1 ligand binding sites or in which the presence or the cellular source of this receptor subtype had previously not been unequivocally established, such as the hippocampus or cerebellar cortex. Moreover, labeled neurons were present in regions not known to receive dopaminergic projections, such as the thalamic and some brainstem nuclei. We conclude that this ISH technique provides a considerable gain in sensitivity and resolution with regard to neurotransmitter receptor mapping.

Use of methacrylate De-embedding protocols for in situ hybridization on semithin plastic sections with multiple detection strategies.

New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing hybridization signal compared to that obtained in other plastics. The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200-400 BPS), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications.

In Situ Hybridization Techniques for Electron Microscopy Detection of Human Papillomavirus Gene Sequences

At present, in situ hybridization (ISH) study of human papillomavirus (HPV) at the ultrastructural level is limited because of the complexity of the techniques. We have developed a method for electron microscopy ISH that is easily adapted for routine use. The method permits the ultrastructural localization of gene sequences of HPV and of any other DNA or RNA sequences, in a technically simple way.CaSki cells containing 400-600 copies of HPV type 16 were grown for 3 days, fixed and processed for embedding in LRW resin at 56°C. An ISH technique was then carried out using biotinylated DNA probes for HPV types 6/11, 16/18, and 31/33/51. Antibiotin antibody conjugated with 15-nm colloidal-gold particles was used to detect the hybrid. All stages of the technique were carried out on slides with wells and on microtiter plates. Monkey kidney cells (BGM) with no evidence of any type of integrated HPV in the genome were used as a negative control.The CaSki cells incubated with the probe specific for HPV types 16/18 yielded a positive intranuclear reaction, manifested as either large accumulations of gold particles located next to the membrane in the nuclear periphery or small groupings (4–8 gold particles) spread throughout the nucleus. No positive reaction was observed in CaSki cells treated with the probe for HPV 6/11 and 31/33/51 nor BGM cells with any of the probes used. (The J Histotechnol 20:337, 1997)

Detection of IgA subclasses and J chain mRNA bearing plasma cells in human dental periapical lesions by in situ hybridization

Humoral immune responses are implicated in the pathogenesis of human dental periapical lesions. To elucidate whether IgA-associated immune systems play a role in the lesions, the in situ hybridization technique was used to detect J chain mRNA expression, which is correlated with the secretion of dimeric IgA. In addition, IgA subclass mRNA-expressing cells were also investigated by double target in situ hybridization (ISH) methodology using digoxigenin- and biotin-labeled IgA subclass specific oligonucleotide probes. This double target ISH technique involved immunochemical detection with an alkaline phosphatase-conjugated antibody and a peroxidase conjugated avidin-biotin complex system to detect IgA subclass mRNA in the formalin-fixed, paraffin wax embedded periapical tissue sections. Twenty-four biopsy samples (16 periapical granulomas and 8 radicular cysts) were examined. IgA subclass mRNA positive plasma cells were detected in all samples. IgA1 mRNA-expressing cells were predominant both in granulomas and cysts (mean = 75.3 +/- 11.2%, 64.8 +/- 21.3%, respectively), and the IgA1 proportion was higher in granulomas than in cysts, although no significant difference was seen between the two lesions (p = 0.132). J chain mRNA positive cells were very sparsely detected in 21/24 cases. The median percentages of J chain mRNA positive cells/IgA mRNA positive plasma cells (4.7%, range 0.3-13.6%) in cysts were significantly higher than in granulomas (1.3%, range 0-7.7%; p = 0.03). This result supports the hypothesis that dimeric IgA may be more actively produced in radicular cysts than in granulomas. These features are thought to reflect the local activation of the periapical immune system in response to environmental factors and indicate that secretory IgA mediated immune defense systems appear to play little role in these lesions. Our results indicate that the IgA-associated immune response in periapical lesions is more similar to serum or systemic IgA responses than to mucosa-associated immune responses where dimeric IgA predominates.

Rapidin situhybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes

In situhybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane hybridization, could be used to decrease hybridization time for ISH. Tissue from swine with an enteric disease produced by a swine coronavirus, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52°C and 70°C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Viral RNA was detected in intestines from as early as 30 min of hybridization by using both buffers with the radiolabelled probe; however, the signal was stronger with the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes. With the REH buffer, hybridization signal intensity was greater at 70°C than at 52°C for both probes. The best results were obtained when small intestinal sections were hybridized at 70°C for 2 h using a radiolabelled or a fluorescein-labelled probe diluted in the REH buffer. The fluorescein-labelled RNA probe with REH buffer resulted in a minimal non-specific signal when compared with the radiolabelled probe. These studies demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should have broad applications in the utilization of probe technology in diagnostics and research for the detection of target ribonucleic acidsin situ

An overview of immunological and genetic methods for detecting swine coronaviruses, transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues.

Transmissible gastroenteritis (TGE) is an enteric disease of swine caused by a coronavirus, designated as transmissible gastroenteritis virus (TGEV). Commonly used methods for TGEV detection include viral isolation and detection of the viral antigen by indirect immunofluorescence (IFA), immunoperoxidase, and immunogold silver staining. Each of these techniques has some advantages and disadvantages. In general IFA and immunohistochemistry are preferred over viral isolation as TGEV isolation is not very reliable because not all field isolates replicate in cell cultures. The diagnosis of TGEV has become more complicated since the emergence of porcine respiratory coronavirus (PRCV). PRCV is believed to be a TGEV mutant, and can not be easily differentiated from TGEV by immunological tests. Nucleic acid probes and polymerase chain reaction (PCR) have successfully been used to detect and differentiate these viruses. These techniques can detect viral nucleic acids in the specimen but do not provide information on the cell types infected by these viruses. Recently we have developed isotopic and nonisotopic in situ hybridization techniques (ISH) for the detection of these viral nucleic acids in formalin-fixed paraffin-embedded tissues. Furthermore, this procedure can differentiate between TGEV- and PRCV-infected cells. By ISH, TGEV is detected in the mature absorptive enterocytes of tissues infected by TGEV and the crypt epithelial cells are also infected but to a lesser extent. For PRCV, the main infected cells are epithelial cells of the bronchioles, type II pneumocytes, and alveolar and septal macrophages. ISH is an excellent tool for studying molecular pathogenesis of these two viruses especially when used in combination with immunohistochemistry.

In situ hybridization forHelicobacter pylori in gastric mucosal biopsy specimens: Quantitative evaluation of test performance in comparison with the clotest and thiazine stain

Numerous detection methods for Helicobacter Pylori (H. pylori) have been developed with varying degrees of purported diagnostic utility. We have developed a rapid nonradioactive in situ hybridization (ISH) method for H. pylori detection in paraffin-embedded tissue and assessed its relative diagnostic performance by receiver operator characteristics (ROC) in comparison to the thiazine stain and CLOtest. Forty-five patients undergoing endoscopy had antral biopsies and concomitant CLOtest performed. ISH for H. pylori was done using a 22-base, biotin-labeled oligonucleotide probe complementary to a portion of H. pylori 16s rRNA with the following sequence: 5'-GGACATAGGCTGATCTTAGC-3'. ISH using this probe was specific for H. pylori with no crossreactivity with other bacterial or fungal organisms. Receiver operator characteristic analysis was used to assess the diagnostic performance of ISH and thiazine techniques. ISH and thiazine stains were done on serial sections, reviewed independently, and scored on a graded scale from 1-5 based upon the degree of assurance of H. pylori presence. Diagnostic performance was assessed in "expert" and "nonexpert" pathologist groups with the CLOtest serving as the invariant test for relative test comparison. The ISH test performed slightly better (ROC area 0.9) than the thiazine (ROC area 0.8) in the nonexpert population, but equally well in the "expert" group (ROC area 0.95, 0.95). ISH followed by routine hematoxylin and eosin staining showed detailed mucosal histology with a dramatic visualization of H. pylori along the surface of the foveolar cells with no evidence of lamina propria invasion. In summary, ISH for H. pylori is an excellent test that is specific, easily read, and allows concomitant detailed histologic mucosal examination.

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa.

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.

Human papillomavirus and Epstein-Barr virus in sinonasal Schneiderian papillomas. An in situ hybridization and polymerase chain reaction study.

Using the polymerase chain reaction (PCR), it has been recently reported that the Epstein-Barr virus (EBV) is present in the majority of Schneiderian sinonasal papillomas (SNP) of the inverted type and may play a role in the pathogenesis of these lesions. The reported prevalence rates of human papillomavirus (HPV) in different types of SNP is also controversial and in need of clarification. Twenty-eight SNP from 27 patients were histologically classified and evaluated for evidence of EBV using PCR and 2 different sensitive and specific in situ hybridization (ISH) procedures for EBER1. Similarly, two methods of ISH were also used for the detection of HPV, using biotinylated DNA probes sensitive for 14 different HPV types as well as more sensitive and specific radioactive RNA probes for HPV types 6, 11, and 16. Polymerase chain reaction was successful in 19 papillomas, including 12 of 19 inverted SNP, 1 of 1 inverted SNP with squamous cell carcinoma, 4 of 5 fungiform SNP, and 2 of 3 oncocytic lesions. Southern blot hybridization of PCR products showed the presence of EBV DNA in two lesions, including one inverted SNP and the single inverted SNP with squamous cell carcinoma. By both DNA- and RNA-mRNA ISH, positivity for EBER was detected in rare stomal lymphocytes but not the overlying epithelium in the inverted SNP with SCC. The remaining cases, including the other inverted SNP positive for EBV by PCR, were completely negative by both ISH techniques. Human papillomavirus was detected by ISH in 1 of 19 (5%) inverted, 1 of 1 (100%) inverted with squamous cancer, 5 of 5 (100%) fungiform, and 0 of 3 (0%) oncocytic SNP. Three SNP contained HPV 6 (all fungiform), three SNP labeled for HPV 11 (two fungiform and the inverted SNP with squamous cancer), and one inverted SNP contained HPV 16. Of the five fungiform SNP, four showed foci of koilocytosis. The results indicate that EBV is not present in sinonasal papillomas. The presence of EBV positive stromal lymphocytes in these lesions may account for a proportion of PCR-positive cases. Oncocytic SNP are unassociated with HPV, whereas inverted SNP contain HPV in a minority of cases. In contrast, fungiform SNP are consistently associated with HPV types 6 and 11 and usually show histologic evidence of viral infection.

Detection of herpes simplex virus (types 1 and 2) and human herpesvirus 6 DNA in human brain tissue by polymerase chain reaction

Background: Previous studies, using a variety of techniques to determine whether herpes simplex virus type 1 (HSV-1) and/or type 2 (HSV-2) are present in normal brains or have a higher incidence in either multiple sclerosis (MS) or psychiatric disorders have yielded conflicting results. Similarly, studies to examine human brain tissue for human herpes virus 6 (HHV-6) have also proved inconsistent. These discrepancies may be partially due to differences in sensitivity of the methods used. Objectives: To determine whether: (i) Herpesvirus latency is a normal occurrence in the human central nervous system (CNS), (ii) the incidence of latency is higher in either demyelinating diseases or schizophrenia (iii) significant virus reactivation occurs in demyelinating diseases. Study Design: Frozen brain tissue from 7 cases of MS/demyelinating disease, 6 cases of schizophrenia and 27 non-neurological and 3 neurological controls were examined by polymerase chain reaction (PCR) for the presence of HSV-1 DNA. Tissue from the above catagories (except schizophrenia) were also examined for HSV-2 and HHV-6 DNA. In situ hybridization (ISH) and immunocytochemistry (ICC) were carried out in formalin-fixed paraffin sections from selected HSV PCR positive cases, including a case of HSV encephalitis (HSE). Results: Cases from all groups were found to be positive for HSV-1 by PCR. Only one case (MS) was found positive for HSV-2, whereas HHV-6 DNA was present in 18 of 23 brains (MS and controls). Only the HSE case gave positive results with ISH and ICC techniques. Conclusions: These results suggest that herpesvirus latency in the human CNS is a common occurrence but there is no obvious correlation with increased incidence in either demyelinating disease or schizophrenia. Furthermore, failure to detect virus by ISH or ICC (except in a case of HSE) indicates lack of any significant virus reactivation in demyelinating diseases.

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization.

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.

Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization.

The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as well as in situ hybridization (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-embedded urinary bladder cancers, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16- or 18-positive signals in all except one of the tumor samples. However, the PCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffin-embedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers.

In situ hybridization detection of cyclin Dl mRNA in centrocytic/mantle cell lymphoma

Centrocytic/mantle cell lymphoma (MCL) is characterized by a specific chromosomal translocation, t(11;14)(q13;q32), which leads to deregulated expression of the G1 cyclin, cyclin D1 (PRAD1, CCND1, BCL1). Cyclin D1 overexpression has been demonstrated in MCL at the mRNA level by Northern blotting and at the protein level by both Western blotting and immunoperoxidase staining. To assess the utility of in situ hybridization (ISH) to detect cyclin D1 mRNA expression in formalin-fixed, paraffin embedded tissue, five MCL specimens from three patients and two cases of B-cell small lymphocytic lymphoma (B-SLL) were studied. BCL1 major translocation cluster gene rearrangements had been previously documented in two MCL patients; the other MCL and the two B-SLL, showed no detectable BCL1 or cyclin D1 rearrangements. ISH was performed using anti-sense 3H-labeled RNA probes for the cyclin D1 3' untranslated region (pPL7) and partial cyclin D1 cDNA (pPL8). ISH experiments using an anti-sense actin RNA probe demonstrated adequate RNA preservation in all cases. Each of five specimens of MCL demonstrated increased cyclin D1 mRNA. In contrast, neither of the two cases of B-SLL demonstrated detectable levels. Overexpression of cyclin D1 mRNA can be detected in MCL by ISH using formalin fixed paraffin embedded tissue. The ISH technique may be useful in diagnosing and classifying low-grade B-cell lymphomas and should be applicable to the study of cyclin D1 mRNA expression in a broad spectrum of lymphoid proliferations and solid tumors.

Topography of forming and resorbing cells on endosteal surfaces of the rabbit humerus by double-staining with in situ hybridization and tartrate-resistant acid phosphatase reaction: a new model to study the bone reaction to loading

Since the first investigations made by Wolff, it has been known that bone adapts to mechanical load. The mechanisms which guide the reaction of osteoblasts and osteoclasts to load are still insufficiently known. In situ hybridization (ISH) allows the detection of intracellular gene transcripts. Therefore, the ISH technique was further developed to allow the detection of pro-alpha 1 (I) collagen gene transcripts on undecalcified bone surfaces. Additionally, this new technique was combined with the tartrate resistant acid phosphatase technique. The combination of the two methods allows the detection of forming and resorbing cells on the same undecalcified bone surface. In addition, a new animal model was developed to study the reaction of bone to mechanical load. This model mimics the situation of bone implants (e.g. hip prosthese), which is a static situation which is dynamically loaded by the action of the patient.

Primary brain lymphoma in acquired immunodeficiency syndrome. Immunophenotype and molecular pathologic characterization in stereotactic biopsy, autopsy and cerebrospinal fluid cytology

In this study we present morphological, cytological, immunophenotypical, and molecular genetic features of ten cases of AIDS-related primary brain lymphomas obtained as sterotactic biopsies, autopsy specimens, or cerebrospinal fluid samples. Histologically, a very characteristic perivascular and diffuse lymphomatous spread in the brain parenchyma was observed. By applying a highly sensitive in situ hybridization technique (ISH) using digoxigenin-labeled antisense riboprobes, abundant Epstein-Barr virus (EBV)-encoded small nuclear RNAs (EBER) transcripts could be demonstrated in each case studied. A combination of ISH with immunohistochemical staining for latent membrane protein (LMP-1) and EBV nuclear antigen-2 (EBNA-2) showed different patterns of EBV latency with a predominance of the broad spectrum (EBER+/EBNA-2+/LMP-1+). Clonal rearrangements of immunoglobulin heavy chain (IgH) genes were demonstrated in four cases using a sensitive polymerase chain reaction. In one patient the rearrangement pattern suggested biclonal lymphoproliferation. Our observations confirm the implication of the EBV in the etiology of AIDS-related primary malignant lymphomas of the brain. The demonstration of EBV gene products and clonal IgH rearrangements even in small biopsy samples is a useful tool for distinguishing between reactive and neoplastic lymphocytic infiltrates, which are frequently observed in brain and cerebrospinal fluid specimens obtained from AIDS patients.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.

Nonrandom numerical aberrations of chromosomes 7, 9, and 10 in DNA-diploid bladder cancer

Double-target in situ hybridization technique (ISH) was applied to 37 cases of bladder cancer to detect numerical aberrations of chromosomes 7, 9, 10, and 11 by centromeric DNA probes. Of 33 evaluable cases, 29 (88%) demonstrated chromosome aberrations. In 17 cases with diploid pattern as measured by flow-cytometric DNA analysis (FCM), 15 (88%) demonstrated chromosome aberrations. Of these, trisomy 7, monosomy 9, and trisomy 10 were detected in three, three, and one case, respectively, as a single chromosome aberration. In 14 (88%) of 16 cases with an aneuploid DNA pattern, chromosome aberrations for two or more chromosomes were demonstrated. A significant correlation was observed between grade of chromosome aberrations and tumor grade ( p < 0.01, Fisher's test), between number of spots for chromosome 7 and peak value in FCM ( p = 0.015, by Spearman rank order test). In eight cases, chromosome aberrations in the tumor were compared with the corresponding pattern in the grossly normal and histologically benign mucosa. Trisomy 10 and monosomy 9 were detected as chromosome numerical aberrations in the histologically normal mucosa, consistent with aberrations in the corresponding patients. We conclude that trisomy 7, monosomy 9, and trisomy 10 may be early events in the evolution of bladder cancer.

Ontogeny of glucocorticoid and D2 receptors in the rat pituitary: an in situ hybridization study

The expression of glucocorticoid and D2 dopamine receptors (GR and D2R) during rat pituitary ontogenesis was studied by in situ hybridization (ISH). On early stages, E13–E14, a weak specific signal for GR mRNA was obvious in the whole Rathke's pouch (RP) whereas subsequently, from E17–E18, strong labelling was restricted to the anterior lobe (AL) and the neural lobe (NL). At the same time, D2R mRNAs appeared in the intermediate lobe (IL) and the long isoform of the D2R (D2R 444) was detectable with specific probes. On the postnatal stages, until adult, GR mRNA, if present, was always undetectable in the IL using the conventional ISH technique. These data indicate a possible early regulation of POMC gene expression by glucocorticoid in corticotrophic cells of the AL and by dopamine in the melanotrophic cells of the IL. The possibility of a negative regulation of GR mRNA by dopamine (DA) in the IL as soon as E17 is discussed.

Double-target in situ hybridization in brightfield microscopy.

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromosome imbalances.

Gamma-aminobutyric acidA-receptor messenger ribonucleic acid (alpha-1 subunit) detection by in situ hybridization.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is most likely involved in the efferent cochlear neurotransmission. In situ hybridization (ISH) results in specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue and allows subsequent visualization of the location of the probe. We used the ISH technique to localize messenger ribonucleic acid (mRNA) sequences of the alpha-1 subunit of the GABAA receptor with an S-35 labeled oligonucleotide probe. Experiments were performed in rat and guinea pig brain sections and surface tissue preparations of the guinea pig cochlea. Positive signals were obtained for the alpha-1 probe in cortical and hippocampal regions of the rat brain and had weaker expression in the guinea pig brain. Alpha-1 subunit mRNA was localized in Purkinje cells and in stellate and basket cells of the stratum moleculare in the rat and guinea pig cerebellum. In surface tissue preparations of the guinea pig cochlea mRNA sequences of the alpha-1 subunit were detectable with high signal expression. Positive signals were seen on both sides of the tunnel of Corti, predominantly in the region of the outer hair cells. The results indicate expression of GABAA-receptor mRNA in cochlear tissue, supporting the importance of GABAA receptors in cochlear neurotransmission.