Recent developments in the use of synthetic oligonucleotides for in situ hybridization histochemistry

Synthetic oligonucleotides have been used with increasing frequency as probes for the detection and study of the regulation of specific mRNAs by in situ hybridization histochemistry. These probes can be easily obtained and used by the nonmolecular biologist, and they have been shown to be effective for the study of a wide range of mRNAs in neuronal and neuroendocrine tissues. Considerations in oligonucleotide probe design, synthesis, purification, and labeling are described in this article, and current procedures for tissue preparation and hybridization are discussed. In addition, control procedures and methods for the quantitation of in situ hybridization by image analysis are discussed. Finally, the combination of this technique with immunocytochemistry and retrograde tract-tracing is reviewed. The coupling of quantitative in situ hybridization with other neuronal markers, e.g., of connectivity, provides an increasingly valuable technology for exploring the regulation of gene expression in a rich anatomical context.     

A method for detection of a cytokine and its mRNA in the central nervous system of the developing rat

We report here an effective and concise method to determine the localization of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, and its mRNA in the central nervous system of pre- and postnatal rats. This method allows for double staining to demonstrate localization of different molecules on the same tissue specimen at the levels of mRNA and proteins by in situ hybridization and immunohistochemistry, respectively. Additionally, the present method gives results more quickly than the conventional isotopic techniques. By use of this method, we carried out immunohistochemistry with an anti-rat MIF polyclonal antibody and demonstrated positive staining using the avidin-biotin complex method (ABC method). To detect its mRNA, we performed nonradioactive in situ hybridization using a digoxigenin (DIG)-labeled RNA probe prepared from a full length fragment of rat MIF cDNA. MIF was strongly expressed in the telencephalon on embryonic day 16. Non-radioactive in situ hybridization with a DIG-labeled RNA probe as well as the immunohistochemistry described here could be applicable to characterize localization of mRNA and proteins of different molecules on the same tissue specimen.     

Rational design of oligonucleotide probes to avoid optimization steps in in situ hybridization

In situ hybridization (ISH) is a widely used technique in neuroscience since it allows a relatively straightforward determination of gene expression in the brain, in respect to distribution as well as in respect to quantification. It is based upon the hybridization of a nucleic acid probe with the mRNA under investigation and does not require the creation of specific antibodies as in immunohistochemistry. However, a major drawback of ISH is the fact that all standard protocols available include time consuming optimization steps of several critical parameters such as tissue fixation, hybridization conditions and washing procedures. Therefore, the aim of our investigation was a rational design of oligonucleotide probes which were adapted to our standard ISH protocol and which could therefore be used without changing any parameter. This approach also worked well for the detection of rare gene products such as neuropeptide receptor mRNAs. To adapt the probes to our standard procedure, sequence, calculated melting temperature, length and secondary structures of the oligonucleotides were considered according to certain constraints as outlined in the following.     

Progressive multifocal leukoencephalopathy: investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe

Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected) showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using this technique. Viral DNA replication may be an important initial step which produces the bizarre "transformed" astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be useful in the diagnosis of PML on brain biopsy specimens.     

JC virus detection by in situ hybridization in brain tissue from elderly patients.

Brains from 10 patients aged 68 to 96 years at time of death were studied for JC viral DNA and common papovaviral capsid protein. In situ hybridization of JC viral DNA was performed by affinity cytochemistry using a biotinylated fragment of JC viral DNA. Immunohistochemistry was performed on brain tissue by the avidin DH-biotinylated horseradish peroxidase technique using polyclonal antibody raised against the papovaviral capsid protein. Viral protein and DNA were detected in 4 of 10 patients. JC virus may be present in the brains of aged patients more frequently than previously suspected.     

The detection of viral nucleic acid sequences in paraffin-embedded subacute sclerosing panencephalitis (SSPE) and progressive multifocal leukoencephalopathy (PML) brain tissues by in situ hybridization

We have been able to detect the viral nucleic acid sequences in formalin-fixed paraffin embedded tissue sections of the brain from SSPE and PML respectively by in situ hybridization using cloned and radiolabelled complementary DNA (cDNA) measles nucleocapsid (NP) and JC virus probes which are gene specific respectively. We have mainly followed in situ hybridization procedure reported by Haase et al, but partly modified the technique including the extensive wash by buffer solution and the elevated concentration of proteinase K. Firstly, to compare with the number of hybridized cells and grain counts, the in situ hybridization were performed in SV 40 infected cells treated with ethanol-acetic acid for 15 minutes or 10% neutral formalin for at least one week to simulate procedure used by pathologists. Similar findings were obtained in both treatment of 1 microgram/ml concentration of proteinase K in ordinary fixed cells and in 100 micrograms/ml in formalin-fixed cells. In addition we investigated the ability of in situ hybridization technique to detect measles in SSPE and papova virus in PML obtained postmortem that were been formalin-fixed paraffin-embedded routinely. In paraffin-embedded SSPE cerebral cortex positive hybridization were obtained in oligodendroglias and neurons using 125I-labelled measles NP probe in 6 days' autoradiographic exposure (Fig. 2 and 3) in our modified technique. Additionally, in PML tissue JC virus nucleic acid sequences were also detected in many oligodendroglias and swollen neurons using 3H-labelled JC virus probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular localization of BM88 mRNA in paraffin-embedded rat brain sections by combined immunohistochemistry and non-radioactive in situ hybridization

With the advent of gene cloning and sequencing, it has become increasingly common to identify novel genes for which no antibody is available. The best approach to study the expression and the distribution of these new genes is by in situ hybridization. One of the challenges with this method is to define the exact cellular subtype where the gene of interest is expressed. Conventional isotopic in situ hybridization methods lack precision for cellular identification because radioactive probes often result in a scattered signal. To identify the exact cellular subtype expressing BM88, we established a rapid colocalization method using non-isotopic in situ hybridization followed by chromogenic immunohistochemistry on the same tissue section. We demonstrated that BM88, which was identified from subtractive hybridization experiments between normal and ischemic tolerant brain tissue, was expressed exclusively in neurons in normal adult rat brain. Paraffin-embedded tissue was used as it resulted in better preservation of tissue and cellular morphology, thus allowing for more accurate histological localization of gene expression. It also allowed for retrospective studies on a number of archived tissue samples.     

Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections

In situ cDNA:mRNA hybridization is a technique that has been developed for the visualization of cDNA:mRNA hybrids in individual cells. To use this technique to answer questions of regulation in heterogeneous populations of cells in the brain, it must be combined with other procedures allowing for the identification of functional subgroups of neurons. We report here a procedure by which in situ cDNA:mRNA hybridization may be combined with retrograde axonal tracing using the fluorescent tracer fast blue. Using this technique, it now becomes possible to measure mRNA regulation in functional subsets of cells defined by their axonal projections.     

In situ polymerase chain reaction demonstration of JC virus in progressive multifocal leukoencephalopathy,including an index case

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease that is caused by JC papovavirus. The virus can be demonstrated in brains with PML using a variety of techniques. In situ polymerase chain reaction (PCR) is a new method that combines the sensitivity of PCR with the histological localization of in situ hybridization. We here show that in situ PCR can detect JC virus in archival tissue sections of 3 cases of PML, including 1 of the original cases described in 1958 and 1 case that was fixed in formalin for 7 weeks. JC virus DNA was amplified directly on tissue and demonstrated in the characteristic enlarged oligodendrocytes and bizarre astrocytes. These data illustrate the utility of the in situ PCR technique in detecting JC virus in archival sections, including those fixed for prolonged periods, and provide a historical footnote to the original report of PML.     

Demonstration of mitochondrial ribosomal RNA in frozen and paraffin–embedded sections of skeletal muscle by in situ hybridization

We have used in situ hybridization with synthetic oligonucleotide probes to the 16S sub–unit of mitochondrial ribosomal RNA to detect mitochondrial accumulations in biopsies of skeletal muscle from 16 patients with mitochondrial myopathies. In all cases, the distribution of the hybridization signal matched the pattern of succinate dehydrogenase activity in adjacent cryostat sections. In situ hybridization also allowed visualization of mitochondrial accumulations in paraffin–embedded sections of skeletal muscle that had been fixed in either De Castro's solution or formalin. DNase pre–treatment did not significantly diminish the intensity of hybridization signal, suggesting that the hybridization is predominantly to RNA. The advantages of this technique are that it allows the investigation of mitochondrial disorders when only paraffin–embedded tissue is available and should also facilitate the demonstration of mitochondrial accumulations in tissues other than skeletal muscle.     

Characterization of proopiomelanocortin mRNA detected by in situ hybridization

In situ hybridization is a method for detecting specific nucleotide sequences by using a labeled complementary nucleic acid probe. We have characterized in situ hybridization in rat pituitary using a cDNA probe directed against a portion of proopiomelanocortin (POMC) mRNA to show its specificity in tissue sections. This characterization includes the size of the target mRNA from tissue processed for in situ hybridization, the dissociation pattern of the cDNA:mRNA hybrid, and quantitation of changes in POMC mRNA levels in the rat pituitary intermediate lobe detected both by in situ hybridization and "dot blot" techniques.     

Decortication and striatal mRNA: increases of mRNA for fibronectin, but not of NCAM or alpha-1 tubulin.

Ipsilateral frontal cortex lesions damaged the corticostriatal input. Northern hybridization analysis showed increases in fibronectin (FN) mRNA, but not changes in mRNA for neural cell adhesion molecule (NCAM), neuro-filament-68 (NF-68) or alpha-tubulin (alpha-1T) 72 h post-lesion. In situ hybridization resolved a different spatial-temporal distribution. The superficial cell layer beneath the wound cavity showed transient elevations of FN mRNA that peaked at 72 h post-lesion. However, in the ipsilateral striatum, FN mRNA was maximal at later times than in the wound cavity, at 240 h post-lesion. Changes in NCAM and alpha-tubulin mRNAs in response to decortication occur only around the wound cavity but not in the deafferentated striatum. The different time courses of mRNA revealed by Northern blot analysis and in situ hybridization are most probably due to contamination of the ipsilateral striatum at dissection with superficial tissue adjacent to the wound cavity. These results suggest that cellular responses to ipsilateral decortication consist of two phases: (i) a wound healing process; and (ii) striatal responses to deafferentation.     

Tyramide signal amplification in brain immunocytochemistry: adaptation to electron microscopy.

The tyramide signal amplification (TSA) technique is well-established in light microscopic immunohistochemistry and in situ hybridization to improve the signal-to-noise ratio. The present study deals with its adaptation to the electron microscopic level using the pre-embedding technique and a modified protocol. The outcome of immunolabeling of most of the antigens tested in brain tissue, including endothelial and neuronal nitric oxide synthase, glial fibrillary acidic protein, and isolectin B4, was greatly improved. If signal amplification is required, the TSA-technique proved to be reliable with high specificity and good ultrastructural resolution.     

Reserpine increases striatal neurotensin mRNA levels.

Reserpine administration has been shown to increase striatal tissue levels of neurotensin with a time course similar to that for striatal dopamine depletion. To determine whether reserpine treatment may increase striatal neurotensin synthesis we have examined striatal neurotensin mRNA levels using in situ hybridization histochemistry. The number of striatal cells expressing neurotensin mRNA was increased 6 h, but not 1 h, after reserpine administration. Thus, the increase in striatal tissue levels of neurotensin after reserpine may be due in part to an increase in peptide synthesis.