In situ hybridization: MODIFICATIONS

Mikael Niku, Dept. Basic Veterinary Sciences, University of Helsinki

Biomedicum 3.4.2006

Todays menu
Tyramide signal amplification Combining ISH and immunohistochemistry Microwave magic Genomic in situ hybridization for tissues PCR in situ hybridization Protocol examples

Traditional nonradioactive ISH

Variations: probe labels

Digoxigenin (DIG, Roche) Most commonly used Detected by high-affinity antibodies Endogenously only in Digitalis plants Biotin Traditional Detected by avidin (very high affinity) Variably present in mammalian tissues 2,4-dinitrophenyl (DNP, PerkinElmer) Comparable to DIG Fluorescent labels (FITC, etc.) May also be detected by antibodies for increased versatility and sensitivity

Variations: enzymes
Alkaline phosphatase
At best, the most sensitive detection system Present in some mammalian tissues Most common substrate: NBT/BCIP (nitroblue tetrazolium / 5-bromo-4-chloro-3-indolyl phosphate), dark blue colour A fluorescent substrate: ELF-97 (Molecular Probes)

Peroxidase
Variably present in mammalian tissues Most common (and most sensitive) substrate: DAB (diaminobenzidine), brown colour Used in tyramide amplification

RNA-ISH AP detection (no tyramides)

RNA-ISH POD detection (with tyramide amp.)

Y-chromosome ISH to bull thymus 1x DIG tyramide + anti-DIG-AP

Y-chromosome ISH to bull thymus 1x biotin-tyramide + avidin-POD

Tyramide signal amplification

(TSA /CSA)

A powerful amplification system Peroxidase-catalyzed label deposition to the target

Peroxidase converts labeled tyramide into a highly reactive radical Tyramide radicals covalently bind to nearby tyrosine residues

Tyramide synthesis
1998)

(Hopman et al.

Tyramide signal amplification

TSA: pros / cons

At best, very effective amplification Covalent binding label not easily lost applications in multistainings, etc. High amplification masks intensity differences Amplifies also background Endogenous peroxidase sometimes a problem Patented & very expensive as commercial kit (but easy to prepare for noncommercial academic use!)
Hopman et al (1998) J Histochem Cytochem 46: 771-777

RNA-ISH to epidermis WITH TYRAMIDE AMPLIFICATION

RNA-ISH to epidermis 6x PROBE CONCENTRATION, NO TYRAMIDE AMPLIFICATION

Y-chromosome ISH to bull brain WITH TYRAMIDE AMPLIFICATION

Y-chromosome ISH to bull brain NO TYRAMIDE AMPLIFICATION

Variations: TSA strategies

Tyramide amplification allows a change from a detection system to another
May use peroxidase first, then go to phosphatase May use one label in the probe, another in the tyramide

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

Optimizing TSA
With a powerful amplification system, any artefacts may also be amplified be extra careful with controls Optimize
Probe concentration (typically 5-10 times less too much probe may sometimes cause loss of signal!) Concentrations of all detection reagents (probably need less, sometimes more) Reaction times (tyramide, colorimetric substrate)

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

Y-chromosome ISH to bull brain 3x tyramide amplification (biotin-tyramide + avidin-POD)

TSA examples
DIG probe anti-DIG-POD DIGtyramide anti-DIG-AP Biotin probe avidin-POD DIGtyramide anti-DIG-AP DIG probe anti-DIG-POD FITCtyramide anti-FITC-FITC DIG probe anti-DIG-POD biotintyramide avidin-POD biotintyramide avidin-POD

Combining ISH and IH

Problem:
In situ protocol destroys several antigens In situ protocol strips / destroys bound detection antibodies Immunohistochemical visualization systems may cause false negatives in ISH (DAB precipitate)

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

The Solution
Use tyramide amplification for immunohistochemistry
Start with IH
Retrieve Detect Amplify with tyramide covalently bound label deposited in tissue

Perform ISH Finish IH

Detect labelled tyramide

IH + ISH

Y-ish + CD45/ML-I. Niku et al. Stem Cells 22(1):12-20.

Microwave magic
Microwave heating speeds up chemical reactions
Exact mechanisms in ISH systems mostly unclear

Try microwaves for everything!

Fixation? Tissue pretreatment! Hybridization?

Photo: Sharp-World

Tissue pretreatment w. microwaves

PFA-fixed paraffin tissues need pretreatments to allow probe penetration Traditionally: protease digestion, HCl incubation
poor reproducibility damage tissue morphology

Microwave heating:
routine in modern immunohistochemistry (antigen retrieval in various buffers) in ISH, stronger signals with better morphology often in combination with mild protease treatment

Y chromosome ISH to PFA-fixed bull thymus WITH MICROWAVE TREATMENT

Y chromosome ISH to PFA-fixed bull thymus NO MICROWAVE TREATMENT

 Kitchen microwave ovens: pulses of constant power; cheap Lab microwave ovens: adjustable power output, exact temperature control; very expensive In any case, standardize your setting:

Optimizing microwave treatments

Effects of buffer

Container Constant volume of buffer Constant number of slides (add mock slides) Constant duration and cooling period (e.g. 15 minutes heating, 20 minutes cooling)

pH 6 citrate buffer most common 2x SSC buffer pH 6

 May speed up & enhance hybridization

e.g. Lan et al. J Histochem Cytochem 44(3):281-287

Microwaves in hybridization

Use low power (for kitchen ovens, add water as buffer for excessive power) Disposable thermometer stickers (electronics industry) help in standardizing the protocol

Genomic ISH
Detection of genetic markers, such as Y chromosomal sequences Sensitivity copy number
Degenerate oligos for high copy number microsatellites

Critical steps:
Efficient permeabilization Denaturation Long enough hybridization (up to 2x o/n)

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

PCR in situ hybridization

Another method of signal amplification PCR amplify the target
labelled or unlabelled nucleotides

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

PCR-ISH: samples & pretreatment

Cryosections or PFA-fixed paraffin stuff
Cryosections: fix in PFA overnight Paraffin: microwave treatment (500 ml 2x SSC pH6, 15 min, 750W)

Both:
Permeabilize: PBS + 1% Tween, 30 min Optimized protK treatment (eg. 0.5 1 g/ml)
Need sufficient permeabilization for reagents, but too much lets PCR products diffuse away

PCR-ISH: PCR reaction

In situ block for PCR machine Prevention of reagent evaporation
Slide sealing chambers

Optimize reagents:
[Mg] different from normal PCR Add BSA to prevent enzyme from sticking to glass 10x polymerase concentration

PCR-ISH: ISH
After PCR, fix sections again Perform normal in situ hybridization to the PCR product Remember good controls!
Negative tissue Reaction without primer Reaction without polymerase

Male, Y primers

Male, no primers

Anna Ekman Dept. Basic Vet. Sci.

Female, Y primers

Examples of protocols
Genomic in situ hybridization to paraffin sections RNA in situ hybridization to cryosections, with tyramide amplification Available online at http://www.vetmed.helsinki.fi/pell/ anatomia/eng/research/protocols/

Protocol example 1: Genomic ISH Samples: 2-4 m paraffin sections of PFA-fixed bovine tissues Probe: 28bp degenerate oligo-DNA
Specific for a highly repetitive Ychromosomal satellite sequence (1000s of targets) Single DIG label

Protocol example 1 / Pretreatments

Deparaffinization (completely!) Microwave heating
3 x 5 min in xylene + rehydration in ethanol series 15 min in 500 ml of 2x SSC pH 6 at 700W, + 20 min cooling at room temp

Packing in Shandon Coverplates Permeabilization: 1% Tween / PBS, 30 min Protease treatment: Protease treatment: 5200 g/ml P6911 (Sigma) in TE buffer pH 7.4, for 30 min at 37C Postfixation: 4% PFA/PBS, 5 min at RT Remove coverplates, dehydrate in ethanol series, air dry

yes yes no some yes yes yes yes yes

Protocol example 1 / Hybridization

Apply hybridization solution
50% formamide, 4x SSPE, ssDNA, Denhardts probe at about 15 nM

Coverslip Heat-denature slides in oven on metal plate (85C 8 min) Pack in airtight containers with some tissue paper moistened with base of hyb solution Hybridize 1-2x overnight at RT

Protocol example 1 / Post-hyb washes Let coverslips drop in 4x SSPE Wash at RT:
4x SSPE / 50% formamide 10 min 4x SSPE rinse 1.5x SSPE 10 min

Transfer to antibody buffer (MAB = maleic acid buffer for AP detection, dont use phosphate buffers here!)

Protocol example 1 / Detection

Back to coverplates Block (Roche blocking reagent in MAB) Incubate in Roche anti-DIG-AP 1:1000 for 2 h Wash Transfer to detection buffer (Tris-HCl pH 9.5, NaCl, MgCl2) Incubate in NBT/BCIP overnight (+4C to RT) Wash Counterstain (hematoxylin) Mount with Faramount

Protocol example 2: RNA-ISH with TSA

Samples: 10 m cryosections of bovine tissues Probes: DIG-labelled riboprobes
0.3 1.3 kb Basic in vitro transcription from high quality linearized plasmid templates 1/3 UTP with 11-DIG-label Purified by LiCl-ethanol precipitation or Sephadex columns Checked by normal agarose gel electrophoresis and Nanodrop concentration measurement

High quality reagents, fresh milliQ water, no DEPC

Protocol example 2 / Pretreatments

Cut fresh cryosections from fairly recent samples Heat at 60C for 1 min, then dry at RT for 1 h Fix: ice-cold 4% PFA in PBS pH 9.5, 1 h (for tyramide amplification, pH 7.4 maybe better) Permeabilize:
0,2 M HCl 6 min 01 g/ml protease K in Tris-Calcium buffer, 30 min at RT no detergents!

Dehydrate in ethanol series, air dry

Protocol example 2 / Hybridization Apply hybridization solution:

buffer like in Y chromosome ISH 20 l original transcription reaction diluted 1:100 1:1000

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

Protocol example 2 / The rest

Wash like genomic ISH, except for the higher temperature (hyb temp) Block in Roche blocking reagent in MAB buffer Incubate with anti-DIG-POD 1:100 for 1 h, wash, transfer to PBS Incubate with DIG-labelled tyramide in PBSimidazole buffer for 10 min Incubate with anti-DIG-AP 1:1000 for 2 h, wash Detect with NBT/BCIP like in genomic ISH

So long, and thanks for all the ISH.