Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.     

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.     

Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.     

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

Summary 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 ofin situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in thein 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 ofin situ hybridization products and tissue antigens.     

A new method to perform nonradioactive tissue printing using digoxigenin-labeled riboprobes

We describe a modified protocol to perform a tissue print northern using digoxigenin-labeled riboprobes. The result of the hybridization is directly visualized on the membrane by an alkaline phosphatase detection system.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.     

Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.

We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.     

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.     

Microwave strategy for improving the simultaneous detection of estrogen receptor and galanin receptor mRNA in the rat hypothalamus.

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor. (J Histochem Cytochem 49:901-910, 2001)     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

Quest for a reliable,valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system

In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentration of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolized the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of³H-and³⁵S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging. Fanally, the procedure permits study of 100% of thin serial sections; hence, alternate sections can be hybridized with sense and antisense riboprobes to detect viral genome and its mRNA or stained, immunocytochemically, to detect viral proteins. Accordingly, it is anticipated that the mechanism of persistent CNS viral infections will be deciphered, at least in part by advances in cytological molecular hybridization.     

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.