Selected In Situ Hybridization Methods: Principles and Application

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

Download Full-text

Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.2.8288860 ◽

1994 ◽

Vol 42 (2) ◽

pp. 149-154 ◽

Cited By ~ 4

Author(s):

I Leger ◽

M Robert-Nicoud ◽

G Brugal

Keyword(s):

In Situ Hybridization ◽

Viral Infections ◽

Imaging Techniques ◽

Simultaneous Detection ◽

Chromosome 1 ◽

Confocal Laser ◽

Nucleolar Proteins ◽

Immunocytochemical Detection ◽

The Individual

The recent application of DNA cloning and non-radioactive in situ hybridization techniques has strengthened the hypothesis of an ordered chromatin structure in interphase nuclei. The arrangement of specific chromosomal regions is not random and is strongly suspected to vary with functional activity. The combination of in situ hybridization and immunocytochemistry, allowing simultaneous detection of nucleic acid sequences and specific antigens in the same nucleus, has already made significant contributions to the study of gene expression, to simultaneous karyotyping and phenotyping of tumor cells, and to in situ analysis of viral infections. This report emphasizes the considerable interest of such combined techniques for functional in situ mapping of the genome at the individual cell level. We propose a method that combines fluorescence immunocytochemical detection of nucleolar proteins and fluorescence in situ hybridization of centromeric and telomeric probes specific for chromosome 1 in two cultured human cell lines. The preparative constraints for a broad application of this procedure are defined so that the cell preparations can be further analyzed by fluorescence microscopic imaging techniques and confocal laser scan microscopy. The two selected sequences of the human chromosome 1 can be localized in the nucleus with respect to nucleolar proteins in a one-step fluorescence microscopic observation.

Download Full-text

RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Methods and Protocols ◽

10.3390/mps4010020 ◽

2021 ◽

Vol 4 (1) ◽

pp. 20

Author(s):

Mujeeb Shittu ◽

Tessa Steenwinkel ◽

William Dion ◽

Nathan Ostlund ◽

Komal Raja ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Expression Patterns ◽

Drosophila Species ◽

Gene Expression Patterns ◽

Probe Design ◽

Tissue Preparation ◽

Design And Synthesis ◽

Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

Download Full-text

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

BMC Ecology and Evolution ◽

10.1186/s12862-021-01939-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Oliver Vöcking ◽

Lucas Leclère ◽

Harald Hausen

Keyword(s):

In Situ Hybridization ◽

Photoreceptor Cells ◽

Visual Cycle ◽

Phylogenetic Distribution ◽

Enzymatic Process ◽

Antibody Staining ◽

The Individual ◽

Functional Components ◽

Visual Rhodopsin

Abstract Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Download Full-text

Intercomparison of four different in-situ techniques for ambient formaldehyde measurements in urban air

Atmospheric Chemistry and Physics Discussions ◽

10.5194/acpd-5-2897-2005 ◽

2005 ◽

Vol 5 (3) ◽

pp. 2897-2945 ◽

Cited By ~ 6

Author(s):

C. Hak ◽

I. Pundt ◽

C. Kern ◽

U. Platt ◽

J. Dommen ◽

...

Keyword(s):

Regression Line ◽

Ambient Air ◽

Urban Site ◽

Optical Absorption Spectroscopy ◽

Measuring Instruments ◽

Chromatographic Technique ◽

In Situ Techniques ◽

Time Period ◽

The Individual

Abstract. Results from an intercomparison of several currently used in-situ techniques for the measurement of atmospheric formaldehyde (CH2O) are presented. The measurements were carried out at Bresso, an urban site in the periphery of Milan (Italy) as part of the FORMAT-I field campaign. Eight instruments were employed by six independent research groups using four different techniques: Differential Optical Absorption Spectroscopy (DOAS), Fourier Transform Infra Red (FTIR) interferometry, the fluorimetric Hantzsch reaction technique (five instruments) and a chromatographic technique employing C18-DNPH-cartridges (2,4-dinitrophenylhydrazine). White type multi-reflection systems were employed for the optical techniques in order to avoid spatial CH2O gradients and ensure the sampling of nearly the same air mass by all instruments. Between 23 and 31 July 2002, up to 13 ppbv of CH2O were observed. The concentrations lay well above the detection limits of all instruments. The formaldehyde concentrations determined with DOAS, FTIR and the Hantzsch instruments were found to agree within ±11%, with the exception of one Hantzsch instrument, which gave systematically higher values. The two hour integrated samples by DNPH yielded up to 25% lower concentrations than the data of the continuously measuring instruments averaged over the same time period. The consistency between the DOAS and the Hantzsch method was better than during previous intercomparisons in ambient air with slopes of the regression line not significantly differing from one. The differences between the individual Hantzsch instruments could be attributed in part to the calibration standards used. Possible systematic errors of the methods are discussed.

Download Full-text

Unobtrusive Observation of Cycling Tourists in the Wild

International Journal of Mobile Human Computer Interaction ◽

10.4018/ijmhci.2014100102 ◽

2014 ◽

Vol 6 (4) ◽

pp. 22-42

Author(s):

Benjamin Poppinga ◽

Martin Pielot ◽

Wilko Heuten ◽

Susanne Boll

Keyword(s):

Field Study ◽

In Situ Observation ◽

In Situ Techniques ◽

In The Wild ◽

The Individual ◽

Post Hoc ◽

Real Challenge ◽

Unobtrusive Observation

The observation of cycling tourists is a real challenge. Traditional in-situ observation techniques fail as they threaten the intimateness of the experience and often interfere with the users' tasks. In post-hoc studies, like interviews, participants are unable to recap all details of their earlier experience accurately. This paper investigates how a hybrid, i.e., in-situ and post-hoc, observation approach can overcome the individual limitations and thereby provide detailed insights without disturbing the cyclists. The authors demonstrate the approach in a field study, where we observed 11 tourists with three unobtrusive in-situ techniques and used the gathered data to jog their memories in a post-hoc interview. They found that the observation technique allows to get detailed and accurate insights, and the communication between experimenter and participant becomes clearer. The authors conclude that hybrid observation would be valuable in other mobile field study settings.

Download Full-text

Variation in highly repetitive DNA composition of heterochromatin in rye studied by fluorescence in situ hybridization

Genome ◽

10.1139/g95-142 ◽

1995 ◽

Vol 38 (6) ◽

pp. 1061-1069 ◽

Cited By ~ 42

Author(s):

A. Cuadrado ◽

N. Jouve ◽

C. Ceoloni

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Repetitive Dna ◽

Dna Sequences ◽

5S Rdna ◽

Individual Identification ◽

Highly Repetitive Dna ◽

The Individual

The molecular characterization of heterochromatin in six lines of rye has been performed using fluorescence in situ hybridization (FISH). The highly repetitive rye DNA sequences pSc 119.2, pSc74, and pSc34, and the probes pTa71 and pSc794 containing the 25S–5.8S–18S rDNA (NOR) and the 5S rDNA multigene families, respectively, were used. This allowed the individual identification of all seven rye chromosomes and most chromosome arms in all lines. All varieties showed similar but not identical patterns. A standard in situ hybridization map was constructed following the nomenclature system recommended for C-bands. All FISH sites observed appeared to correspond well with C-band locations, but not all C-banding sites coincided with hybridization sites of the repetitive DNA probes used. Quantitative and qualitative differences between different varieties were found for in situ hybridization response at corresponding sites. Variation between plants and even between homologous chromosomes of the same plant was found in open-pollinated lines. In inbred lines, the in situ pattern of the homologues was practically identical and no variation between plants was detected. The observed quantitative and qualitative differences are consistent with a corresponding variation for C-bands detected both within and between cultivars.Key words: fluorescence in situ hybridization, repetitive DNA, rye, Secale cereale, polymorphism.

Download Full-text

Searching for ROS1 Rearrangements in Lung Cancer by Fluorescent In Situ Hybridization: The Importance of Probe Design

Journal of Thoracic Oncology ◽

10.1097/jto.0000000000000605 ◽

2015 ◽

Vol 10 (8) ◽

pp. e83-e85 ◽

Cited By ~ 2

Author(s):

Arnaud Uguen ◽

Pascale Marcorelles ◽

Marc De Braekeleer

Keyword(s):

Lung Cancer ◽

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Probe Design

Download Full-text

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.1.1729352 ◽

1992 ◽

Vol 40 (1) ◽

pp. 135-141 ◽

Cited By ~ 16

Author(s):

E J Speel ◽

B Schutte ◽

F C Ramaekers ◽

A H Hopman

Keyword(s):

In Situ Hybridization ◽

Network Formation ◽

Transitional Cell ◽

Fluorescein Isothiocyanate ◽

Staining Technique ◽

Dna Probe ◽

Detection Systems ◽

Amplification Method ◽

The Individual

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.

Download Full-text

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

Canadian Journal of Microbiology ◽

10.1139/m96-136 ◽

1996 ◽

Vol 42 (10) ◽

pp. 1061-1071 ◽

Cited By ~ 39

Author(s):

Marc E. Frischer ◽

Peter J. Floriani ◽

Sandra A. Nierzwicki-Bauer

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Differential Sensitivity ◽

Probe Design ◽

Oligonucleotide Probes ◽

Whole Cells ◽

Gene Probes ◽

Wide Range

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [γ-32P] ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.Key words: 16S rRNA, oligonucleotide probes, in situ hybridization.

Download Full-text