In situ HybridizationMuhammad Noman Naseem

D.V.M, M.Phil. (Pathology)

Cellular And Molecular Pathology

nomannaseem97@yahoo.com

Mob# +92-3226302788

INTRODUCTION

• In situ hybridization was invented by Joseph G. Gall.

• In suite hybridization is biological assay (like ELISA, PCR) for molecular diagnosis.

• In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain information about gene expression and genetic loci.

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In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g. plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells and in circulating tumor cells (CTCs).

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• In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insights into physiological processes and disease pathogenesis.

• However, in situ hybridization requires that many steps be taken with precise optimization for each tissue examined and for each probe used.

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• In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation and function of genes.

Applications of ISH

• Prenatal test during pregnancy

• Conventional prenatal tests for chromosomal abnormalities such as Down Syndrome rely on analyzing the number and appearance of the chromosomes the karyotype.

• Molecular diagnostics tests such as microarray comparative genomic hybridisationtest a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive.

Application Continue……• Treatment;• Pharmacogenomics• Some of a patient's s—slight differences in their

DNA—can help predict how quickly they will metabolize particular drugs; this is called pharmacogenomics.

• For example, the enzyme CYP2C19 metabolizes several drugs, such as the anti-clotting agent Clopidogrel, into their active forms. Some patients possess polymorphisms in specific places on the 2C19 gene that make poor metabolisers of those drugs; physicians can test for these polymorphisms and find out whether the drugs will be fully effective for that patient

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• Pathogenomics

• Molecular diagnostics are used to identify infectious diseases such as chlamydia, influenza virus and tuberculosis; or specific strains such as H1N1 virus.

Types of ISH• Today there are two basic ways to visualize your

RNA and DNA targets in situ—• fluorescence (FISH) and • chromogenic (CISH) detection. • Characteristics inherent in each method of

detection have made FISH and CISH useful for very distinct applications. While both use a labeled, target-specific probe that is hybridized with the sample, the instrumentation used to visualize the samples is different for each method.

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• Chromogenic in situ hybridization (CISH) enables you to gain genetic information in the context of tissue morphology.

• Fluorescence in situ hybridization (FISH) enables you to assay multiple targets simultaneously and visualize co-localization within a single specimen.

In Situ Hybridization Protocol

General procedure and tips for in situ hybridization using antibody detection.

In situ hybridization indicates the localization of gene expression in their cellular environment. A labeled RNA or DNA probe can be used to hybridize to a known target mRNA or DNA sequence within a sample.

This labeled RNA or DNA probe can then be detected by using an antibody to detect the label on the probe. The probes can therefore be used to detect expression of a gene of interest and the location of the mRNA.

How To Store a Sample• Preserving DNA is easy because it is a highly

stable molecule.

• However, preserving RNA is much more difficult due to presence of RNase enzyme. This may be found on glassware, in reagents and on the operator and their clothing.

• RNase will quickly destroy any RNA in the cell or the RNA probe itself.

• Therefore, users must ensure they use sterile techniques, gloves, and solutions to prevent RNase from contaminating and destroying the probe or tissue RNA.

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• General sample storage when using frozen sections:

• For good results on older slides, the slides should not be stored dry at room temperature. They should be stored either in 100% ethanol at -20°C, or in a plastic box covered in saran wrap at -20°C or -80°C. Slides stored in this way can be used for several years.

Choice of probe

• RNA probes:RNA probes should be between 250 to 1500 bases in length. Probes approximately 800 bases long exhibit the highest sensitivity and specificity.

• DNA probes:DNA probes can also provide high sensitivity for in situ hybridization.. However, they do not hybridize as strongly to the target mRNA molecules as RNA probes.

DIG (Digoxigenin) labeled RNA probe In situ hybridization protocol

• The protocol shown here describes the use of DIG labeled single stranded RNA probes to detect expression of the gene of interest in paraffin embedded sections.

• It is a highly sensitive technique. Step (1) Deparaffinization• If using formaldehyde fixed paraffin embedded sections.• Before proceeding with the staining protocol, the slides

must be deparaffinized and rehydrated. • Incomplete removal of paraffin can cause poor staining of

the section

CONTINUE…..• Place the slides in a rack, and perform the following washes:

1) Xylene: 2 x 3 min2 ) Xylene 1:1 with 100% ethanol: 3 min3 ) 100% ethanol: 2 x 3 min4 ) 95% ethanol: 3 min5 ) 70 % ethanol: 3 min6 ) 50 % ethanol: 3 min7 ) Running cold tap water to rinse

• Keep the slides in the tap water until ready to perform antigen retrieval.

• t no time from this point onwards should the slides be allowed to dry.

• Drying out will cause non-specific antibody binding and therefore high background staining.

ISH Protocol Continue…. Step(2) Antigen retrieval • (antigen retrieval A process in which tissues are heated with a microwave oven,

steam (pressure) cooker, autoclave or ultrasound, which causes antigens that are difficult or impossible to stain in formalin-fixed paraffin sections of tissue to become readily stainable by standard immunohistochemical methods.)

• Digest with 20 µg/ml proteinase K in pre-warmed 50 mMTris for 10 to 20 min at 37°C. The time of incubation and concentration of proteinase K may require some optimization.

• The concentration of proteinase K and the incubation time for this step will require optimization. We recommend trying a proteinase K titration experiment to determine the optimal conditions. Insufficient digestion will result in a reduced hybridization signal. Over digestion will result in poor tissue morphology, making localization of the hybridization signal very difficult. The concentration of proteinase K needed will vary depending upon the tissue type, length of fixation, and size of tissue.

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Step(3) Rinse slides 5 times in distilled waterStep(4) Immerse slides in ice cold 20% (v/v)

acetic acid for 20 sec. This will permeabilize the cells to allow access to the probe and the antibody.

Step(5) Dehydrate the slides by washing for approximately 1 min each wash in 70% ethanol, 95% ethanol and 100% ethanol then air dry.

Step(6) Add 100 µl of hybridization solution to each slide.

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Step(7) Incubate the slides for 1 hr in a humidified hybridization chamber at the desired hybridization temperature. Typical hybridization temperatures range between 55 and 62°C .(The optimal hybridization temperature for the probe depends on the percentage of bases present in the target sequence. The concentration of cytosine and guanine in the sequence are an important factor)

Step(8) Dilute the probes in hybridization solution in PCR tubes. Heat at 95°C for 2 min in a PCR block. This will denature the RNA or DNA probe. Chill on ice immediately to prevent reannealling.

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Step(8) Drain off the hybridization solution. Add 50 to 100 μl of diluted probe per section (ensure the entire sample is covered). Incubate in the humidified hybridization chamber at 65°C overnight. While incubating, the sample on the slide can be covered with a cover slip to prevent evaporation.

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Step(9) Stringency washes:Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e. only exact sequence matches will remain bound).

• This step removes non-specific and/or repetitive DNA / RNA hybridization. The less concentrated the salt solution and the longer the duration of the wash and the temperature, the higher the stringency and the more DNA / RNA will be removed

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• . Step(10) Wash twice in MABT (maleic acid buffer containing Tween 20) for 30 min at room temperature.

• MABT is gentler than PBS and is more suitable for nucleic acid detection

• Step(11) Dry the slides.

• Step(12) Transfer to a humidified chamber and add 200 µl blocking buffer to each section (MABT + 2% BSA, milk or serum). Block for 1 to 2 hours, at room temperature.

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Step(13) Drain off the blocking buffer. Add the anti-‘label’ antibody at the required dilution in blocking buffer. Check the antibody datasheet for a recommended concentration/dilution. Incubate for one to two hours at room temperature.

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Step(14) Wash slides 5 times with MABT, 10 min for each wash, at room temperature.

Step(15) Wash the slides 2 times for 10 min each at room temperature with pre-staining buffer.(100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2).

Step(16) Fluorescence – please proceed to step 18Other – return slides to humidified chamber and follow manufacturer’s instructions for color development.

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Step(17) Fluorescence ……….Other – return slides to humidified chamber and follow manufacturer’s instructions for color development.

Step(18) Rinse slides in distilled water.

Step(19) Air dry the slides for around 30 min. Wash in 100% ethanol, then air dry thoroughly.

Step(20) Mount using DePeX mounting solution.