In situ hybridization:MODIFICATIONS

Mikael Niku, Dept. Basic Veterinary Mikael Niku, Dept. Basic Veterinary Sciences, Sciences,

University of HelsinkiUniversity of Helsinki

Biomedicum 3.4.2006Biomedicum 3.4.2006

Today’s menu• Tyramide signal amplificationTyramide signal amplification• Combining ISH and Combining ISH and

immunohistochemistryimmunohistochemistry• Microwave magicMicrowave magic• Genomic in situ hybridization for Genomic in situ hybridization for

tissuestissues• PCR in situ hybridizationPCR in situ hybridization• Protocol examplesProtocol examples

Traditional nonradioactive ISH

Variations: probe labels• Digoxigenin (DIG, Roche)Digoxigenin (DIG, Roche)

– Most commonly usedMost commonly used– Detected by high-affinity antibodiesDetected by high-affinity antibodies– Endogenously only in Endogenously only in Digitalis Digitalis

plantsplants• BiotinBiotin

– TraditionalTraditional– Detected by avidin (very high Detected by avidin (very high

affinity)affinity)– Variably present in mammalian Variably present in mammalian

tissuestissues• 2,4-dinitrophenyl (DNP, PerkinElmer)2,4-dinitrophenyl (DNP, PerkinElmer)

– Comparable to DIGComparable to DIG• Fluorescent labels (FITC, etc.)Fluorescent labels (FITC, etc.)

– May also be detected by antibodies May also be detected by antibodies for increased versatility and for increased versatility and sensitivitysensitivity

Variations: enzymes• Alkaline phosphataseAlkaline phosphatase

– At best, the most sensitive detection systemAt best, the most sensitive detection system– Present in some mammalian tissuesPresent in some mammalian tissues– Most common substrate: NBT/BCIP (nitroblue Most common substrate: NBT/BCIP (nitroblue

tetrazolium / 5-bromo-4-chloro-3-indolyl tetrazolium / 5-bromo-4-chloro-3-indolyl phosphate), dark blue colourphosphate), dark blue colour

– A fluorescent substrate: ELF-97 (Molecular A fluorescent substrate: ELF-97 (Molecular Probes)Probes)

• PeroxidasePeroxidase– Variably present in mammalian tissuesVariably present in mammalian tissues– Most common (and most sensitive) substrate: Most common (and most sensitive) substrate:

DAB (diaminobenzidine), brown colourDAB (diaminobenzidine), brown colour– Used in tyramide amplificationUsed in tyramide amplification

RNA-ISHAP detection (no tyramides)

RNA-ISHPOD detection (with tyramide amp.)

Y-chromosome ISH to bull thymus1x DIG tyramide + anti-DIG-AP

Y-chromosome ISH to bull thymus1x biotin-tyramide + avidin-POD

Tyramide signal amplification (TSA /CSA)

• A powerful amplification systemA powerful amplification system• Peroxidase-catalyzed label Peroxidase-catalyzed label

deposition to the targetdeposition to the target– Peroxidase converts labeled tyramide Peroxidase converts labeled tyramide

into a highly reactive radicalinto a highly reactive radical– Tyramide radicals covalently bind to Tyramide radicals covalently bind to

nearby tyrosine residuesnearby tyrosine residues

Tyramide synthesis (Hopman et al. 1998)

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TSA: pros / cons• At best, very effective amplificationAt best, very effective amplification• Covalent binding Covalent binding label not easily lost label not easily lost

applications in multistainings, etc.applications in multistainings, etc.• High amplification masks intensity High amplification masks intensity

differencesdifferences• Amplifies also backgroundAmplifies also background• Endogenous peroxidase sometimes a Endogenous peroxidase sometimes a

problemproblem• Patented & very expensive as commercial kit Patented & very expensive as commercial kit

(but easy to prepare for noncommercial (but easy to prepare for noncommercial academic use!)academic use!)– Hopman et al (1998) J Histochem Cytochem 46: Hopman et al (1998) J Histochem Cytochem 46:

771-777771-777

RNA-ISH to epidermisWITH TYRAMIDE AMPLIFICATION

RNA-ISH to epidermis6x PROBE CONCENTRATION,

NO TYRAMIDE AMPLIFICATION

Y-chromosome ISH to bull brainWITH TYRAMIDE AMPLIFICATION

Y-chromosome ISH to bull brainNO TYRAMIDE AMPLIFICATION

Variations: TSA strategies• Tyramide amplification allows a Tyramide amplification allows a

change from a detection system to change from a detection system to anotheranother– May use peroxidase first, then go to May use peroxidase first, then go to

phosphatasephosphatase– May use one label in the probe, another May use one label in the probe, another

in the tyramidein the tyramide

• Tyramide amplification may be used Tyramide amplification may be used repeatedly repeatedly still more signal, be still more signal, be careful with background & artefactscareful with background & artefacts

Optimizing TSA• With a powerful amplification With a powerful amplification

system, any artefacts may also be system, any artefacts may also be amplifiedamplified be extra careful with controls be extra careful with controls

• OptimizeOptimize– Probe concentration (typically 5-10 Probe concentration (typically 5-10

times less – too much probe may times less – too much probe may sometimes cause sometimes cause lossloss of signal!) of signal!)

– Concentrations of all detection reagents Concentrations of all detection reagents (probably need less, sometimes more)(probably need less, sometimes more)

– Reaction times (tyramide, colorimetric Reaction times (tyramide, colorimetric substrate)substrate)

Tyramide amplified RNA-ISH: UNSPECIFIC STAINING (endogenous POD, etc)

Y-chromosome ISH to bull brain3x tyramide amplification (biotin-tyramide + avidin-POD)

TSA examples• DIG probe DIG probe anti-DIG-POD anti-DIG-POD DIG- DIG-

tyramide tyramide anti-DIG-AP anti-DIG-AP• Biotin probe Biotin probe avidin-POD avidin-POD DIG- DIG-

tyramide tyramide anti-DIG-AP anti-DIG-AP• DIG probe DIG probe anti-DIG-POD anti-DIG-POD FITC- FITC-

tyramide tyramide anti-FITC-FITC anti-FITC-FITC• DIG probe DIG probe anti-DIG-POD anti-DIG-POD

biotin-tyramide biotin-tyramide avidin-POD avidin-POD biotin-tyramide biotin-tyramide avidin-POD avidin-POD

Combining ISH and IH• Problem:Problem:

– In situ protocol destroys several In situ protocol destroys several antigensantigens

– In situ protocol strips / destroys In situ protocol strips / destroys bound detection antibodiesbound detection antibodies

– Immunohistochemical visualization Immunohistochemical visualization systems may cause false negatives in systems may cause false negatives in ISH (DAB precipitate)ISH (DAB precipitate)

• Thus, need to detect antigen Thus, need to detect antigen before ISH, but visualize afterbefore ISH, but visualize after

The Solution• Use tyramide amplification for Use tyramide amplification for

immunohistochemistryimmunohistochemistry– Start with IHStart with IH

• RetrieveRetrieve• DetectDetect• Amplify with tyramide Amplify with tyramide covalently covalently

bound label deposited in tissuebound label deposited in tissue

– Perform ISHPerform ISH– Finish IHFinish IH

• Detect labelled tyramideDetect labelled tyramide

IH +

IS

H

Y-ish +

CD

45/ML

-I. Niku et al. Stem

Cells 22(1):12-20.

Microwave magic• Microwave heating speeds up chemical Microwave heating speeds up chemical

reactionsreactions– Exact mechanisms in ISH systems mostly Exact mechanisms in ISH systems mostly

unclearunclear

• Try microwaves for everything!Try microwaves for everything!– Fixation?Fixation?– Tissue pretreatment!Tissue pretreatment!– Hybridization?Hybridization?

Photo: Sharp-World

Tissue pretreatment w. microwaves

• PFA-fixed paraffin tissues need PFA-fixed paraffin tissues need pretreatments to allow probe penetrationpretreatments to allow probe penetration

• Traditionally: protease digestion, HCl Traditionally: protease digestion, HCl incubationincubation– poor reproducibilitypoor reproducibility– damage tissue morphologydamage tissue morphology

• Microwave heating: Microwave heating: – routine in modern immunohistochemistry routine in modern immunohistochemistry

(antigen retrieval in various buffers)(antigen retrieval in various buffers)– in ISH, stronger signals with better morphologyin ISH, stronger signals with better morphology– often in combination with mild protease often in combination with mild protease

treatmenttreatment

Y chromosome ISH to PFA-fixed bull thymusWITH MICROWAVE TREATMENT

Y chromosome ISH to PFA-fixed bull thymusNO MICROWAVE TREATMENT

Optimizing microwave treatments

• Kitchen microwave ovens: pulses of Kitchen microwave ovens: pulses of constant power; cheapconstant power; cheap

• Lab microwave ovens: adjustable power Lab microwave ovens: adjustable power output, exact temperature control; very output, exact temperature control; very expensiveexpensive

• In any case, standardize your setting:In any case, standardize your setting:– ContainerContainer– Constant volume of bufferConstant volume of buffer– Constant number of slides (add mock slides)Constant number of slides (add mock slides)– Constant duration and cooling period (e.g. 15 Constant duration and cooling period (e.g. 15

minutes heating, 20 minutes cooling)minutes heating, 20 minutes cooling)• Effects of bufferEffects of buffer

– pH 6 citrate buffer most commonpH 6 citrate buffer most common– 2x SSC buffer pH 62x SSC buffer pH 6

Microwaves in hybridization

• May speed up & enhance May speed up & enhance hybridizationhybridization– e.g. Lan et al. J Histochem Cytochem e.g. Lan et al. J Histochem Cytochem

44(3):281-28744(3):281-287

• Use low power (for kitchen ovens, Use low power (for kitchen ovens, add water as buffer for excessive add water as buffer for excessive power)power)

• Disposable thermometer stickers Disposable thermometer stickers (electronics industry) help in (electronics industry) help in standardizing the protocolstandardizing the protocol

Genomic ISH• Detection of genetic markers, such Detection of genetic markers, such

as Y chromosomal sequencesas Y chromosomal sequences• Sensitivity – copy numberSensitivity – copy number

– Degenerate oligos for high copy number Degenerate oligos for high copy number microsatellitesmicrosatellites

• Critical steps:Critical steps:– Efficient ”permeabilization”Efficient ”permeabilization”– DenaturationDenaturation– Long enough hybridization (up to 2x o/n)Long enough hybridization (up to 2x o/n)

A-B. Male, Y probe. C-D. Male, Y probe + 10x unlabeled probe. E-F. Female, Y probe. Niku et al. Dev Comp Immunol 26(8): 689-95.

PCR in situ hybridization• Another method of signal Another method of signal

amplificationamplification• PCR amplify the targetPCR amplify the target

– labelled or unlabelled nucleotideslabelled or unlabelled nucleotides

• For better specificity, use For better specificity, use unlabelled PCR product and detect unlabelled PCR product and detect with ISHwith ISH

PCR-ISH: samples & pretreatment

• Cryosections or PFA-fixed paraffin stuffCryosections or PFA-fixed paraffin stuff– Cryosections: fix in PFA overnightCryosections: fix in PFA overnight– Paraffin: microwave treatment (500 ml 2x Paraffin: microwave treatment (500 ml 2x

SSC pH6, 15 min, 750W)SSC pH6, 15 min, 750W)

• Both:Both:– Permeabilize: PBS + 1% Tween, 30 minPermeabilize: PBS + 1% Tween, 30 min– Optimized protK treatment (eg. 0.5 – 1 Optimized protK treatment (eg. 0.5 – 1

g/ml)g/ml)• Need sufficient permeabilization for reagents, Need sufficient permeabilization for reagents,

but too much lets PCR products diffuse awaybut too much lets PCR products diffuse away

PCR-ISH: PCR reaction• In situ block for PCR machineIn situ block for PCR machine• Prevention of reagent evaporationPrevention of reagent evaporation

– Slide sealing chambersSlide sealing chambers

• Optimize reagents:Optimize reagents:– [Mg] different from normal PCR[Mg] different from normal PCR– Add BSA to prevent enzyme from sticking to Add BSA to prevent enzyme from sticking to

glassglass– 10x polymerase concentration10x polymerase concentration

PCR-ISH: ISH• After PCR, fix sections againAfter PCR, fix sections again• Perform normal in situ Perform normal in situ

hybridization to the PCR producthybridization to the PCR product

• Remember good controls!Remember good controls!– Negative tissueNegative tissue– Reaction without primerReaction without primer– Reaction without polymeraseReaction without polymerase

Anna Ekman

Dept. Basic Vet. Sci.

Male, Y primers

Female, Y primers

Male, no primers

Examples of protocols• Genomic in situ hybridization to Genomic in situ hybridization to

paraffin sectionsparaffin sections• RNA in situ hybridization to RNA in situ hybridization to

cryosections, with tyramide cryosections, with tyramide amplificationamplification

Available online atAvailable online at

http://www.vetmed.helsinki.fi/pell/http://www.vetmed.helsinki.fi/pell/anatomia/eng/research/protocols/anatomia/eng/research/protocols/

Protocol example 1: Genomic ISH

• Samples: 2-4 Samples: 2-4 m paraffin sections m paraffin sections of PFA-fixed bovine tissuesof PFA-fixed bovine tissues

• Probe: 28bp degenerate oligo-DNAProbe: 28bp degenerate oligo-DNA– Specific for a highly repetitive Y-Specific for a highly repetitive Y-

chromosomal satellite sequence chromosomal satellite sequence (1000s of targets)(1000s of targets)

– Single DIG labelSingle DIG label

Protocol example 1 / Pretreatments

• Deparaffinization (completely!)Deparaffinization (completely!)– 3 x 5 min in xylene + rehydration in ethanol 3 x 5 min in xylene + rehydration in ethanol

seriesseries• Microwave heatingMicrowave heating

– 15 min in 500 ml of 2x SSC pH 6 at 700W, + 15 min in 500 ml of 2x SSC pH 6 at 700W, + 20 min cooling at room temp20 min cooling at room temp

• Packing in Shandon CoverplatesPacking in Shandon Coverplates• Permeabilization: 1% Tween / PBS, 30 minPermeabilization: 1% Tween / PBS, 30 min• Protease treatment: Protease treatment: Protease treatment: Protease treatment:

5-200 5-200 g/ml P6911 (Sigma) in TE buffer g/ml P6911 (Sigma) in TE buffer pH 7.4, for 30 min at 37pH 7.4, for 30 min at 37CC

• Postfixation: 4% PFA/PBS, 5 min at RTPostfixation: 4% PFA/PBS, 5 min at RT• Remove coverplates, dehydrate in ethanol Remove coverplates, dehydrate in ethanol

series, air dryseries, air dry

yes

yes

yesno

yesyesyesyes

some

Protocol example 1 / Hybridization

• Apply hybridization solutionApply hybridization solution– 50% formamide, 4x SSPE, ssDNA, Denhardts50% formamide, 4x SSPE, ssDNA, Denhardts– probe at about 15 nMprobe at about 15 nM

• CoverslipCoverslip• Heat-denature slides in oven on metal Heat-denature slides in oven on metal

plate (85plate (85C 8 min)C 8 min)• Pack in airtight containers with some Pack in airtight containers with some

tissue paper moistened with base of hyb tissue paper moistened with base of hyb solutionsolution

• Hybridize 1-2x overnight at RTHybridize 1-2x overnight at RT

Protocol example 1 / Post-hyb washes

• Let coverslips drop in 4x SSPELet coverslips drop in 4x SSPE• Wash at RT:Wash at RT:

– 4x SSPE / 50% formamide 10 min4x SSPE / 50% formamide 10 min– 4x SSPE rinse4x SSPE rinse– 1.5x SSPE 10 min1.5x SSPE 10 min

• Transfer to antibody buffer (MAB = Transfer to antibody buffer (MAB = maleic acid buffer for AP detection, maleic acid buffer for AP detection, don’t use phosphate buffers here!)don’t use phosphate buffers here!)

Protocol example 1 / Detection

• Back to coverplatesBack to coverplates• Block (Roche blocking reagent in MAB)Block (Roche blocking reagent in MAB)• Incubate in Roche anti-DIG-AP 1:1000 for 2 hIncubate in Roche anti-DIG-AP 1:1000 for 2 h• WashWash• Transfer to detection buffer (Tris-HCl pH 9.5, Transfer to detection buffer (Tris-HCl pH 9.5,

NaCl, MgClNaCl, MgCl22))• Incubate in NBT/BCIP overnight (+4Incubate in NBT/BCIP overnight (+4C to RT)C to RT)• WashWash• Counterstain (hematoxylin)Counterstain (hematoxylin)• Mount with FaramountMount with Faramount

Protocol example 2: RNA-ISH with TSA

• Samples: 10 Samples: 10 m cryosections of bovine tissuesm cryosections of bovine tissues• Probes: DIG-labelled riboprobesProbes: DIG-labelled riboprobes

– 0.3 – 1.3 kb0.3 – 1.3 kb– Basic in vitro transcription from high quality Basic in vitro transcription from high quality

linearized plasmid templateslinearized plasmid templates– 1/3 UTP with 11-DIG-label1/3 UTP with 11-DIG-label– Purified by LiCl-ethanol precipitation or Sephadex Purified by LiCl-ethanol precipitation or Sephadex

columnscolumns– Checked by normal agarose gel electrophoresis and Checked by normal agarose gel electrophoresis and

Nanodrop concentration measurementNanodrop concentration measurement

• High quality reagents, fresh milliQ water, no High quality reagents, fresh milliQ water, no DEPCDEPC

Protocol example 2 / Pretreatments

• Cut fresh cryosections from fairly recent Cut fresh cryosections from fairly recent samplessamples

• Heat at 60Heat at 60C for 1 min, then dry at RT for 1 hC for 1 min, then dry at RT for 1 h• Fix: ice-cold 4% PFA in PBS pH 9.5, 1 h (for Fix: ice-cold 4% PFA in PBS pH 9.5, 1 h (for

tyramide amplification, pH 7.4 maybe tyramide amplification, pH 7.4 maybe better)better)

• Permeabilize:Permeabilize:– 0,2 M HCl 6 min0,2 M HCl 6 min– 0–1 0–1 g/ml protease K in Tris-Calcium buffer, 30 g/ml protease K in Tris-Calcium buffer, 30

min at RTmin at RT– no detergents!no detergents!

• Dehydrate in ethanol series, air dryDehydrate in ethanol series, air dry

Protocol example 2 / Hybridization

• Apply hybridization solution:Apply hybridization solution:– buffer like in Y chromosome ISHbuffer like in Y chromosome ISH– 20 20 l original transcription reaction l original transcription reaction

diluted 1:100 – 1:1000diluted 1:100 – 1:1000

• Pack in airtight boxes + humidifierPack in airtight boxes + humidifier• Incubate at 50-55Incubate at 50-55C overnightC overnight

Protocol example 2 / The rest

• Wash like genomic ISH, except for the Wash like genomic ISH, except for the higher temperature (hyb temp)higher temperature (hyb temp)

• Block in Roche blocking reagent in MAB Block in Roche blocking reagent in MAB bufferbuffer

• Incubate with anti-DIG-POD 1:100 for 1 h, Incubate with anti-DIG-POD 1:100 for 1 h, wash, transfer to PBSwash, transfer to PBS

• Incubate with DIG-labelled tyramide in PBS-Incubate with DIG-labelled tyramide in PBS-imidazole buffer for 10 minimidazole buffer for 10 min

• Incubate with anti-DIG-AP 1:1000 for 2 h, Incubate with anti-DIG-AP 1:1000 for 2 h, washwash

• Detect with NBT/BCIP like in genomic ISHDetect with NBT/BCIP like in genomic ISH

So long,and thanks for all the ISH.