In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.     

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 5. Inhibition of RNA Synthesis in Mesophyll Cells

The synthesis of mesophyll cell RNA in tobacco leaf tissue infiltrated with the incompatible bacterium Pseudomonas pisi was investigated by light and electron microscope autoradiography of H³-uridine uptake. Interpretation of the light microscope quantitative data was complicated by the oscillation in levels of cytoplasmic RNA synthesis that resulted from the physical process of fluid infiltration (seen in the control tissue). Direct comparison with the control showed that the presence of bacteria resulted in a rapid decrease in the synthesis of host cell RNA. This effect was clear within the first 1½h (induction period) of the hypersensitive reaction, where it was particularly marked in the cytoplasm of palisade cells. Electron microscope autoradiography showed that the presence of bacteria did not cause complete cessation of RNA synthesis in mitochondria and chloroplasts. The early inhibition of RNA synthesis preceeded fine structural (degenerative) change, and should be regarded as one of the primary events associated with the induction of host cell necrosis.     

Radioimagers as an alternative to film autoradiography forin situ quantitative analysis of125I-ligand receptor binding and pharmacological studies

Summary Three radioimagers, the μ-imager, the β-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacologicalin situ quantitative analysis. Two iodinated ligands¹²⁵I-interleukin-1α and¹²⁵I-gonadotropin releasing hormone agonist, were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical γ detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the μ-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and β-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion forin situ quantitative studies on tissue sections. They combine precise imaging forin situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, broughtin situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.     

A simple procedure for obtaining autoradiographs of G-banded chromosomes

Chromosomal preparations from cells flash-labeled with ³H-dT were Giemsa-banded following trypsin digestion, allowed to air-dry, and then were coated with a layer of 1% Formvar. The slides were subsequently coated with radiographic emulsion (NTB2) and processed for autoradiography. The resulting chromosomes had distinct G-banding and radiographic labeling patterns. Chemographic grain formation in the emulsion, normally caused by Giemsa stain, was prevented by the film of Formvar, allowing a very low background grain level.     

Autoradiography of Mobile,C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions

Summary Attempts to investigate the cellular localization of keratin mRNAs byin situ hybridization with specific [³⁵S]-labelled cDNA probes to mouse epithelia have been seriously impeded by the uncontrollable detachment of frozen tissue sections on conventionally coated glass slides (i.e. those coated with egg white, gelatine, collagen). Similarly, a variety of other coating and attachment devices have proved to be unsatisfactory or impracticable for large scale investigations. These difficulties were completely overcome andin situ hybridization was possible after a short immersion of the glass slides in a 2% solution of 3-aminopropyltriethoxysilane in acetone. This treatment provides the glass surface with aminoalkyl groups which are apparently able to react covalently with aldehyde or ketone functions of frozen tissue sections. The resulting firm adhesion of the sections enabled us to investigate the influence of different fixation and prehybridization procedures on the quality of thein situ hybridization. It was found that especially harsh prehybridization, involving hydrochloric acid, heat and proteinase K treatment, drastically reduces the morphological integrity of the sections, thus rendering a reliable assignment of the label difficult. In contrast, a mild prehybridization, consisting mainly of a rehydration of the sections in phosphate-buffered saline and equilibration in 0.1 M glycine, leaves the morphology intact and leads to a highly efficient and specificin situ hybridization.     

Origin and Kinetics of Resident Tissue Macrophages

Abstract To elucidate the origin and renewal kinetics of peritoneal macrophages, as a typical example of the mononuclear phagocytic system, syngeneic rats were treated with tritiated thymidine [³H]TdR and leucocytes were transferred to unlabelled recipients over a bilateral arteriovenous shunt. Labelled and unlabelled monocytes were evenly distributed in both animals as shown by autoradiography. It was ascertained that no ‘autoradiographically’ detectable reutilization of label occurred and that transferred cells showed undisturbed kinetics. the results imply: 1 resident peritoneal macrophages derive from blood monocytes; 2 peritoneal macrophages represent a homogeneous population in respect to their cellular origin; 3 blood monocytes as a myelogeneous cell line do not represent a generative end cell. They migrate into the tissue (peritoneal cavity) and differentiate into resident macrophages, undergoing on average one mitosis per cell during a period of approximately 7 days. 4 resident peritoneal macrophages are derived 50% from blood monocytes and 50% from division in situ; and 5 under steady-state conditions the renewal rate amounts to 0. 18%/h, which yields a half-life time of 16 days and a renewal time of 23 days.     

Tissue localization of [125I]triiodothyronine in the periorbital area of mice: a microautoradiographic study

A significant retention of [¹²⁵I]triiodothyronine ([¹²⁵I]T₃) in the retrobulbar orbital area of mice has been previously shown. The present study was initiated to determine tissue and intracellular localization of the thyroid hormone in the above area which is concerned in human Graves' disease of the thyroid.Male and female Balb C mice were intravenously injected with 0.1 mL of [¹²⁵I]T₃ (0.2 mCi/gmg). At various time intervals (30 s-10 min) the animals were sacrificed, bled and periorbital tissues were isolated under a dissecting microscope. Three series of samples were prepared: (a) frozen samples for cryomicrotome sections, (b) samples fixed in 10% formaldehyde for paraffin embedded tissues and (c) samples fixed in paraformaldehyde (2%), glutaldehyde (2%) and 0.1 M sodium cacodylate for embedding in Epon-Araldite-DDSA. Sections 5 μ m and 400–600 Å thick for light and electron microscopy, respectively, were coated with Ilford L4 emulsion and exposed for 9–21 days. Light microscope autoradiography demonstrated that [¹²⁵I]T₃ injected intravenously is rapidly transported in the cells of fat tissue of the peribulbar orbital area and tissues with glandular or muscular function: the hormone showed a high affinity for the intra- and extraorbital lacrymal gland cells, the cells of the Harder's gland, those of the sebaceous and meibomian glands of the eye-lids, as well as for local muscular structures. Electron microscope autoradiography showed that radioactivity is already localized inside the cells 30 s after the i.v. injection of [¹²⁵I]T₃ and it is distributed throughout the cytoplasm, with a higher concentration in the vesicles of the Harder's gland cells (rich in lipids and porphyrin), in the endoplasmic reticulum and the mitochondria of the lacrymal glands. 10 min after injection, a shifting of the radioactivity towards the nucleus area was observed. In conclusion, after _vivo injection, the thyroid hormone rapidly penetrates the cells of fat glandular and muscular tissues in the orbital area. Intracellularly, the affinity of the hormone for the secretory vesicles, rough endoplasmic reticulum, mitochondria and nucleus suggest that T₃ could play a role in secretory and metabolic functions of the tissues in the retrobulbar orbital area.     

Multi-substrate chromosome preparations for high throughput comparative FISH

Background  

A modification of a standard method of fluorescence in situ hybridisation (FISH) is described, by which a combination of several substrates and probes on single microscope slides enables more accurate comparisons of the distribution and abundance of chromosomal sequences and improves the relatively low throughput of standard FISH methods.     

AUTORADIOGRAPHIC DETERMINATION OF S35 IN TISSUES AFTER INJECTION OF METHIONINE-S35 AND SODIUM SULFATE-S35

The loss of "bound" S³⁵ that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S³⁵ or Na₂S³⁵O₄. Valid autoradiography of bound S³⁵ in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S³⁵ is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S³⁵ given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S³⁵.     

Localization of the 5S RNA genes in Zea mays by RNA-DNA hybridization in situ

5S RNA was extracted from Zea mays tissue and iodinated in vitro with ¹²⁵I to a high specific activity. Acrylamide gel electrophoresis of the ¹²⁵I-5S RNA, 11/2 weeks after iodination demonstrated that most of the 5S RNA molecules were degraded to half-size or smaller. In situ hybridization with this iodinated RNA to pachytene microsporocyte chromosomes showed that the 5S RNA cistrons are located near the end of the long arm of chromosome 2. No obvious association of the 5S locus with the nucleolus was seen during pachytene or later stages.     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

High resolution electron microscope autoradiography on negative stained specimens

Summary A new method for applying the electron microscope autoradiography to negatively stained specimens is presented.Free ribosomes and ribosome crystals, labeled with ³H-uridine, have been isolated from the rat liver and from the whole hypothermic chick embryo, respectively. The samples, fixed and negatively stained with uranyl acetate, have been treated for autoradiography with the Kodak NTE nuclear emulsion.This method allows to obtain autoradiograms characterized by a very high resolution power of well stained specimens.The perspectives of this new field of ultrastructural investigation are discussed.     

An autoradiographic study of prostaglandin E1 and/or its metabolites in mouse kidney tissue

The use of grain density autoradiography was evaluated as a means of quantifying the distribution of injected prostaglandin E₁ and/or its metabolites in the mouse kidney. Fifty microcuries of ³H-PGE₁ were injected into each of three mice which were sacrificed at 20 min, 40 min and 45 h after injection. Sections of frozen kidneys were mounted on prepared liquid emulsion slides. Following appropriate exposure and processing, the radioactivity in various regions was quantified by grain counting using an eyepiece grid superimposed over the field at 1000X. The highest grain density was found over the papilla and the second highest grain density occurred at the region of the inner zone of the cortex. In addition, the percentage decrease of grain density from tissue taken 40 min after injection when compared to that from tissue taken 20 min after injection implied that these two regions retained the radioactivity more so than the outer zone of the cortex, the outer zone of the medulla and the glomeruli.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models

Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5′ and 3′ probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.