section in situ hybridization - university of helsinki · 2008-04-18 · section in situ...

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Section in situhybridization

Kirsi Sainio

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Section / cellular ISHtypes

Radioactive ISH for cells and tissuesections – radiolabeling of probes anddetection by autoradiographyNon-radioactive ISH – probes labeledwith haptens or fluorochromes –cellular, chromosomal or tissue sectionISH

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Sections

Paraffin/resin embeddeds sectionsFrozen sectionsVibratome sectionsElectron microscopy samples

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Optimizing ISH

Optimized ISH for section (as well as wholemount) protocols share several commongoals:- retention of tissue morphology- rendering tissue permeable to probe- retaining target mRNA within the tissue- effective penetration and binding ofprobes- reduction of nonspecific background

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Optimizing ISH

The critical parameters that result insuccessful ISH are type of fixative andlength of tissue fixation, method forembedding fixed tissue, agents usedfor sample permeabilization, choice ofhybridization conditions, and post-hybridization treatment

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Fixation

Perfusion is much better at preserving tissuequality and RNA integrity because of therapid spread of fixative through the cellsIn addition, perfusion results in ISH datawith low background due to clearance ofblood cells from the tissueFixation by immersion, on the other hand,should be used when perfusion is notpossible - for example with clinical samplesor embryonic tissues

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Fixatives

Fixation should ideally prevent the lossof cellular RNAs during hybridizationwhile preserving accessibility of thetarget RNA to the probePrecipitating fixatives (such asethanol/acetic acid or Carnoy'sSolution) function by precipitatingproteins to trap the RNA inside cells

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Fixatives

They provide the best probepenetrationTissues fixed by precipitating fixativesare subject to loss of target mRNA andthe cell’s morphological structure(Lawrence and Singer, 1985), resultingin poor ISH data quality

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Fixatives

The primary fixative of choice of mostinvestigators is 4% neutral bufferedformalin or 4 % paraformaldehydeAldehyde fixatives are not always thebest alternative although it seems thatthey tend to be the ONLY alternative

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Fixatives

Tissue fixation by formaldehyde worksby crosslinking amino groups, therebypreventing loss of the mRNA targetDuring hybridization, high temperatureand formamide remove some of thesecrosslinks

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Fixatives

This promotes penetration of theprobe, but may also lead to unwantedloss of the target RNAThus, the ratio between thetemperature of hybridization and thestrength of fixation is very importantto obtain an optimal signal

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Fixatives

When using RNA probes thehybridization temperature should behigh enough to ensure specific bindingof the probeFixation of the tissue under alkaline pHsometimes dramatically improves thesignal when using RNA probes

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Embedding

Cryostat sections of frozen tissue andparaffin embedded tissue sectionshave both been effectively used forISHIn general, paraffin-embedded tissuesshow better morphology than frozentissueParaffin embedding requires moretissue processing and can result inRNA loss and low ISH signal (Pintarand Lugo, 1985)

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Embedding

Paraffin sections should be used withcaution for ISH experiments onmammalian tissues where sensitivity iscriticalparaffin sections still have particularvalue in preparation of clinic,pathological and research samples forlong-term protection of tissuemorphology

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Permeabilization

The most critical step in successfull ISHboth in sections and in whole mountsUsually enzymatic (proteinase K) orchemical (HCl) permeabilizationDifferent samples require differenttreatments!!For instance brain tissues fixed in 4%paraformaldehyde overnight: deproteinationby PK is either unnecessary or detrimentalto RNA retention

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Permeabilization

PK digestion of the cell may result inloss of mRNAs or a loss of morphologyaddition of HCl diluted intriethanolamine increases detectionsensitivity in paraformaldehyde fixedsamples, possibly due to its ability todenature ribosomes, thus exposingadditional target mRNAs to probe

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Specificity

In sections background signal arisesprimarily from nonspecific retention of probein tissue sections (due to electrostaticinteractions between probe and tissuemacromolecules)

Several chemical functional groups inproteins (such as amine and carboxylategroups) are believed to induce thisnonspecific binding

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Specificity

Minimize this source of background bytreating tissue slides with acetic anhydrideand triethanolamine (Hayashi et al., 1978)Acetylation of amine groups by aceticanhydride, routinely used in ISH protocols,maybe important in reducing backgrounds(for probes larger than 2.0 kb) (Lawrenceand Singer, 1985)

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Specificity

Another way to decrease nonspecific probebinding is to saturate the binding sites onproteins by incubating tissue withprehybridization solutionficoll, bovine serum albumin, polyvinylpyrrolidone, and nucleic acidscompete with the nonspecific binding ofprobes to tissueHowever, addition of the above reagents tothe hybridization buffer does not completelyprevent background signal

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Specificity

Nuclease treatment after hybridization is stillnecessary for reducing this nonspecificsignal (nuclease treatment degradesunhybridized, single stranded probe)Without RNase treatment, the backgroundwith [33P]-labeled RNA probes may be sohigh that specific hybridization signal is notdiscernableRNA probes tend to exhibit high levels ofnonspecific binding, so RNase treatmentcould help if this is a problem

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Specificity

High stingency washing conditionsafter cRNA-mRNA ISH decrease thebackgroundMostly washes away the unboundnucleotides and off-target hybridsMay also affect somewhat the specificbinding

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SpecificityWhile there are different recipes for makinghybridization buffers, the inclusion ofdextran sulphate in the hybridizationsolution increases probe binding to targetmRNAincluding 10% dextran sulphate enhancesISH signal several foldtoo much dextran sulphate in thehybridization buffer will induce highbackground, which is difficult to remove inpost hybridization washes

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Probes

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Labels

Radioactive methods are sensitive, butrequire radionucleotides, are time-consuming and give poor detection incellular level (autoradiographydetection)Demanding method, but once set-upworks fairly constantly and gives goodresults

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LabelsNon-radioactive methods are also sensitive insection level, give more possibilities in the choiceof label, are quick, give good resolution in singlecell level, give a possibility to double-labelling oreven combination of ISH andimmunohistochemistryEqually demanding method, sometimes difficultto detect small amount of targetGIVES THE DETECTION IN SINGLE CELL LEVEL

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Labels

Radiolabels:– For RNA ISH S35-labeled UTP most often

used, also P33 can be used– S35 labelled RNA probes usually give

higher backgrounds– dithiothreitol (DTT) should be added to all

solutions used in prehybridization,hybridization, and posthybridizationwashes

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Radioactive ISH

Detection possible only byautoradiographyIf this is not done properly, it can spilthe whole ISH!Based on ”standard” photographyemulsion/development processTakes several days/weeks

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Radiolabeled probes

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Radioactive ISH

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Analysis of the results

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How to visualize autoradiography ?

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Photoshop helps …

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… to make it look like a realthing

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Artificial colors to visualizeautoradiography

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Radioactive ISH

When sensitive method is neededTime is sometimes money!Not suitable for high-throughputstudiesMore hazardous waiste productsAutoradiography is difficult and canspoil the whole thing…

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Non-radioactive ISHNonisotopic labeling systems (such asdigoxigenin and biotin) are also frequently usedfor section ISH studiesSame labels and detection methods than inwhole mount ISHPossibility to multiple labelings and modificationsPossibility to include proteinimmunohistochemistyFaster, high throughput studies are possibleAutomated systems possible

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MULTI ISH/immunohistochemistry

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Non-radioactive sectionISH

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When it is nice, it is nice…

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Rmpr

5 days 1 month

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Artificial coloring can beapplied also here

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Different haptens anddetection methods can beused

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Automated section ISH

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Automated section ISH

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Reliable results

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Cellular level detection

Kirsi SainioBiokemia ja KehitysbiologiaBiolääketieteen laitos

Power and pitfalls

Reliable, fast, easy to use, gives goodresultsOptimization possible and easyRelatively expensive, sometimes doesnot give any detection without anyobvious reasonStill worth trying!

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Clinical applications

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FISHybridization

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FISH

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FISH in cells

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FISH in isolatedchromosomes