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doi: 10.1177/41.12.8245419pmid: 8245419
In situ hybridization provides invaluable information regarding the localization of gene expression in heterogeneous tissues. The technique is extremely sensitive and can detect the amount of mRNA contained in a single cell. This review provides a starting point for those who wish to begin using in situ hybridization in their own laboratories. The procedure outlined here is based on 35S-labeled riboprobes and has been used with many probes and tissues with a greater than 90% success rate on the first hybridization. The importance of appropriate controls is stressed. Clusters of silver grains after hybridization do not necessarily indicate specific mRNA localization. Regions of the tissue rich in nuclei often appear to cause spurious binding of probes and have high backgrounds often mistaken as positive signals. The most difficult aspect of in situ hybridization is not to get clusters of silver grains on the slide but rather to do the appropriate controlled experiments to ensure that the signal is real and is not due to some artifactual binding of the probe to the tissue.
Stahl, W L; Eakin, T J; Baskin, D G
doi: 10.1177/41.12.8245420pmid: 8245420
In this article we discuss strategies for selecting oligonucleotides to target isoform-specific mRNAs, drawing on our experience with isotopically labeled oligonucleotides for ISH of Na,K-ATPase mRNA alpha- and beta-subunit isoforms. Oligonucleotide probes based on one of these isoforms have a high probability of forming nonspecific hybrids with related isoform mRNAs. The design and selection of isoform-specific ISH and how their nucleotide structure influences hybridization are reviewed, as well as basic principles in identifying and evaluating candidate probes. Controls such as Tm analysis and GC content are evaluated. For distinguishing among multiple isoforms of gene families, choose lowest possible homology between isoforms consistent with other factors that influence probe performance.
Miller, M A; Kolb, P E; Raskind, M A
doi: 10.1177/41.12.8245421pmid: 8245421
We present a simple, reliable method for simultaneous detection of two distinct mRNAs within the same tissue sample by double in situ hybridization histochemistry. Sections are hybridized with a cocktail of radiolabeled and digoxigenin-labeled cRNA probes. The digoxigenin-labeled probe is detected with an alkaline phosphatase (AP)-dependent chromogen reaction and then the radiolabeled probe is detected by conventional autoradiography. The sensitivities of the two detection methods are comparable and demonstrate the feasibility of using double in situ hybridization histochemistry to investigate the co-localization and co-regulation of mRNAs that are expressed at relatively low levels.
doi: 10.1177/41.12.8245422pmid: 8245422
Biotinylated probes for in situ hybridization (ISH) are now widely used to detect RNAs and viral genomes at the light and electron microscopic levels. Many protocols for ISH with biotinylated probes are now available. The most critical parameters that influence sensitivity and specificity of the procedure are the nature of the probe, the mode of fixation or incorporation of biotin, and the mode of detection of biotin. Biotin can be detected with antibodies or with avidin (or streptavidin) to which is coupled a marker that can be identified under the microscope. Here we review the protocols, pitfalls, advantages, and disadvantages of biotinylated probes for ISH.
Brooks, P J; Kaplitt, M G; Kleopoulos, S P; Funabashi, T; Mobbs, C V; Pfaff, D W
doi: 10.1177/41.12.8245424pmid: 8245424
We describe a procedure for detection of low-abundance cellular RNAs by in situ hybridization histochemistry, using single-stranded DNA probes produced by amplified primer extension labeling with Taq polymerase. We have used this approach to detect a number of high- and low-abundance RNA species and have found it to be a simple and reproducible method of obtaining sensitive probes for in situ hybridization studies. For example, DNA probes generated by amplified primer extension labeling can detect low-abundance heteronuclear RNAs in individual neurons. Since this procedure does not involve recombinant DNA technology or microbiological facilities, it should prove useful to a wide variety of investigators studying the regulation of gene expression at the cellular level.
doi: 10.1177/41.12.8245425pmid: 8245425
The goal of quantitative autoradiography (QAR) in in situ hybridization (ISH) is to determine the amount of radioactive oligonucleotide or riboprobe present in the corresponding area of the tissue slice that produced an autoradiographic image. This article discusses (a) some of the considerations related to selection and use of computer image analysis systems for accomplishing this objective, (b) development of 14C plastic autoradiographic standards for ISH QAR with 33P, (c) using QAR to develop Tm curves for ISH probes, and (d) measurement of resolution with video imaging systems for QAR.
doi: 10.1177/41.12.8245426pmid: 8245426
We describe a new stereological method for analyzing data derived from the in situ hybridization procedure. This method should prove important, since data summarization in terms of grains per anatomic area by sampling of tissue sections may lead to faulty interpretations. Using computer simulation of measurements taken from a two- and a three-dimensional perspective, we show how the detection of molecular changes can be influenced by multiple structural events. Cell volume, the volume of the structure, and the number of labeled cells in an anatomic structure are all important parameters that must be assessed to obtain accurate results. We then outline in detail a simple and efficient method for estimation of the number of mRNA molecules in cells and in the total structure. By estimation of the abundance of mRNA molecules in three dimensions, we can make better inferences concerning gene expression in a structural context.
Canitrot, Y; Lautier, D; Lahmy, S; Vigo, J; Viallet, P; Salmon, J M
doi: 10.1177/41.12.8245427pmid: 8245427
Simultaneous study of intracellular quantification and distribution of fluorescent probes is difficult when cell staining is not homogeneous. This occurs after mitochondrial staining with rhodamine 123 (R123). Classical techniques for evaluation of intracellular R123 fluorescence, such as flow cytometry, are based on measurement of the global fluorescence intensity but do not take into account parameters that reflecting cellular distribution of the probe. For simultaneously studying intracellular quantification and distribution of R123 with fluorescence image analysis, we delineated a mask of the cell, generated from a fluorescent image of the plasma membrane stained by nile red (NR). After a preliminary study of the fluorescence characteristics of R123 and NR to avoid artifacts and optimize conditions of staining, quantification and distribution of intracellular R123 studies were performed by superimposition of the mask on the R123 fluorescence image. This protocol was applied to leukemic cells and allowed estimation of individual cell parameters such as mean fluorescence intensity and standard deviation, the latter providing information of the cellular distribution of R123. Moreover, it permitted demonstration of the redistribution of R123 in the whole cell when coincubated in the presence of nigericin.
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