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See the difference with TSA—the ultimate technology for unparalleleddetection sensitivity

TSA Signal Amplification (TSA) Systems

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Increase your immunohistochemistry and in situ hybridization detection capabilities

with PerkinElmer’s proprietary Tyramide Signal Amplification technology (TSA™)—

and see clearly why TSA is the standard for superior IHC and ISH sensitivity in

labs around the world.

The remarkable sensitivity enhancements that TSA delivers let you see previously

undetectable levels of protein or nucleic acid—for invaluable new information and

insight into your research. Hundreds of technical publications to date provide proof

of the sensitivity, versatility and applicability of TSA technology to a wide range of

scientific research fields. Add TSA to your protocols and you, too, will

see the difference.

For unparalled sensitivity,

TSA technology

Tyramide Signal Amplification is a patented* technology invented by PerkinElmer scientists that amplifies bothchromogenic and fluorescent signals in many applications. TSA can be used in any application that allows theaddition of horseradish peroxidase (HRP) into its protocol, such as immunohistochemistry, in situ hybridization,ELISA and microarray-based differential gene and protein expression studies. In standard IHC and ISH protocols,use of TSA results in a significant increase in sensitivity, without loss of resolution or increase in background.

PerkinElmer is the world’s sole source of patented TSA tech

*TSA is protected by US patents: 5,731,158, 5,583,001, and 5,196,306 and foreign equivalents.

www.perkinelmer.com 3

makes all the difference

Quick Glance

TSA is easily integrated into any protocol after aninitial addition of horseradish peroxidase (HRP).The HRP is used to catalyze the deposition andbinding of a labeled (e.g., biotin, DNP or otherlabeling moieties) tyramide onto tissue sectionsor cell preparations previously blocked with proteins. This reaction is quick (less than 10minutes). The binding is covalent because thereaction intermediately dimerizes with tyrosineresidues on the surface-bound endogenous proteins. These labels can then be detected by standard chromogenic or fluorescent tech-niques. Since the added labels are only deposit-ed proximal to the enzyme site, there is minimal,if any, loss of resolution.

Diagram of TSA direct fluorescence detection.

hnology— the standard for superior IHC and ISH sensitivity.

Innovative amplification technology maintains signal clarity

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Increase Sensitivity

Maximizing the sensitivity of methods aimed at thecellular localization of proteins and nucleic acids is of particular importance when target levels are known orsuspected to be low. Addition of TSA to your protocolmay reveal localized targets previously undetectableand therefore unsuspected — without loss of resolutionor increase in background.

See the difference in your

Do Multi-Target Detection

TSA systems have been designed to allow utilizationof various chromogens or fluorophores. This flexibilityfacilitates applications in which multi-target detectionin the same sample is of interest.

TSA can provide numerous benefits when you add it to your immunohistochemistry or

Gene mapping of c-myc,exon 2 gene (855 bp)mapped to chromosome15, band D2, mousemetaphase chromosomes.Sensitivity improvementwith TSA allows use ofsmaller (cDNA, oligos)probes for more preciselocalization. Detection of single copy genomicsequence was achievedusing a probe less than 2 kb.

Conserve Antibody or Probe

Application of TSA may be beneficial even when anincrease in sensitivity is not required because TSAallows use of less antibody or probe. Typically, thesame level of sensitivity can be achieved with a significant reduction in the amount of target antibody(up to 1,000-fold) or probe used.

IHC of EBV antigen in Hodgkin’s Lymphoma of mixed cellularity.Comparison shows (A) Anti-EBV dilution 1:25, without TSAenhancement, (B) Anti-EBV dilution 1:25,000, enhanced by TSA.(Courtesy of R. Von Wasielewski and S. Gignac, PathologischesInstitut de Medizinischen Hochscule, Hannover, Germany.)

A

B

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Multi-target detectionenables simultaneousvisualization of multiple targets.

CHR 15 D2

Eliminate PCR

TSA is simpler and faster than IS-PCR with equivalentsensitivity. It detects even low or single copy genes. No special equipment is required.

Detection of HIV-1 DNA in formaldehyde-fixed lymph node tissues from positive autopsy material. (A) In situ PCR, 20 cycles. (B) TSA-enhanced ISH. (Courtesy of Dr. J. Luka and C. Afflerbach,Eastern Virginia Medical School, Norfolk, VA.)


research with TSA in your protocol

in situ hybridization protocols. Use of TSA is particularly valuable when you need to:

Obtain High Resolution

TSA provides exquisite resolution— for clearer imagesthan other methods.

Comparison of direct immunofluorescent staining and TSA-enhanced direct fluorescent staining of CMV infected cells. (A) Direct fluorescent staining. (B) Direct fluorescent stainingenhanced by TSA.

Reduce Interference

Biotin-free TSA Plus DNP Systems provide a simpleprotocol and eliminate biotin quenching procedures.Biotin-free formats also eliminate background problems.

Detection of p53 mRNA in lung tissue. Digoxigenin-labeled p53RNA probes were hybridized to paraffin-embedded lung tissue.Comparison shows (A) standard digoxigenin detection with alkaline phophatase/BCIP-NBT (60 minute substrate incubation),and (B) TSA Plus DNP with alkaline phosphatase/BCIP-NBT (15 minute substrate incubation).

Get Faster Results

See results within a day versus up to a week forradiometric detection, and achieve equivalent sensitivity and resolution.

Comparison of radiometric and TSA-amplified ISH detectionof muscarinic receptor mRNA in rat brain hippocampus. M-1oligo-nucleotide probes were labeled with 33P and fluorescein-N6-ddATP by 3' end-labeling and were hybridized to frozen ratbrain tissue sections. (A) Autoradiography of 33P was carriedout for one month. (B) TSA amplification with DAB.

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B

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Immunohistochemistry

TSA Direct Fluorescence Detecti

HRP

SA

BLOCKING REAGENT

1˚ Ab

2˚ Ab

HRP

1˚ Ab

2˚ Ab

SA TT

B

B

T

F

T

F

T

F

T

F

T

F

T

F

T

F

F

F

TARGETTARGET

Detection of Target

Signal Amplification

Visualization via Fluorescence Microscopy

Amplification Actual Timeand Visualization Added toProcedures Protocol

Blocking Step 30 min

Wash Step 3 x 5 min

Incubation offluorophore tyramide 3–10 min

Wash Step 3 x 5 min

Production of ReactiveTyramide by HRP

Key:

B = BiotinF = FluorophoreT = TyramideSA = StreptavidinHRP = Horseradish PeroxidaseAP = Alkaline Phosphatase

TSA Plus Cyanine 3. Dilution 1:10,000,000

Conventional detection withCyanine 3 conjugated 2˚ antibody.Dilution 1:1,000

Standard TSA Cyanine 3. Dilution 1:100,000

Mouse Brain, 20x magnification, 2 second exposure


In Situ Hybridization

PROBE

TARGET

BLOCKING REAGENT

T

F

T

F

T

F

T

F

T

F

HRP

SA

B

HRP

SA

B

T

F

T

F


Visualization via Fluorescence Microscopy

Detection of Target

Key:

B = BiotinF = FluorophoreT = TyramideSA = StreptavidinHRP = Horseradish PeroxidaseAP = Alkaline PhosphataseD = Digoxigenin



Wash Step 3 x 5 min

Incubation offluorophore tyramide 3–10 min

Wash Step 3 x 5 min

Production of ReactiveTyramideby HRP

Embryonic mouse brain histone H4 mRNA ISH, using TSAPlus Cyanine 3 andMAP2 immunoreactivity(IHC) with TSA PlusFluorescein System.

on

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TSA Indirect Chromogenic or Fluo

(Biotin-based or Biotin-free)Immunohistochemistry



Wash Step 3 x 5 min

Incubation ofbiotinyl tyramide 3–10 min

Wash Step 3 x 5 min

Addition of enzyme conjugate 30 min

Wash Step 3 x 5 min

Incubation of chromogen or fluorophore

HRP

SA

HRP

1˚ Ab

2˚ Ab

1˚ Ab

2˚ Ab

HRP

BLOCKING REAGENT

1˚ Ab

2˚ Ab

SA

SA

B

B

TB

T T

B

T

B

T

B

T

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T

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HRP

SA

HRP

SA

HR

P

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T

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HRP

SA

HRP

SA

HRP

SA

SA

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T

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T

T

B

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T

B

BHRP

HRP

SA

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HRP

1˚ Ab

2˚ Ab

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HRP

SA

HRP

SA

HR

P

SA

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HRP

SA

HRP

SA

HRP

SA

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T

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B

B

THRP

SA

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TARGETTARGET

HRP

SA

SA


Detection of Amplified Signal

Visualization


Detection of Target



Visualization

PROBE

TARGET

BLOCKING REAGENT

T

B

T

B

T

B

T

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T

B

HRP

SA

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HRP

SA

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T

B

HRP

SA

HRPSA

HR

PSA

HR

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SAHRP

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HRP

SA

HRP

SAT

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BB

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HRP

SA

HRP

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SAHRP

SA

HRP

SA

HRP

SAT

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T

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T

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BB

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B


Detection of Amplified Signal

Detection of Target Amplification Actual Timeand Visualization Added toProcedures Protocol


Wash Step 3 x 5 min

Incubation ofbiotinyl tyramide 3– 10 min

Wash Step 3 x 5 min

Addition of enzyme conjugate 30 min

Wash Step 3 x 5 min

Incubation of chromogen orfluorophore


rescence Detection

(Biotin-based or Biotin-free)

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Visualization Options for Indirect

Immunohistochemistry

Amplification and Visualization Procedures

After signal amplification, the deposited biotin can be directly visualized by any of the following methods:

Addition of SA-HRP followed by horseradish peroxidase catalyzedchromogen

Addition of SA-AP followed byalkaline phosphatase catalyzed chromogen

Addition of SA-Fluorophore(SA-Fluorescein)(SA-Coumarin)(SA-Texas Red®)

viewed by fluorescence microscopy

SA

HRP

1˚ Ab

2˚ Ab

T

B

T

B

HRP

SA

HRP

SA

HR

P

SA

T

B

T

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HRP

SA

HRP

SA

HRP

SA

B

T

B

T

T

B

B

THRP

SA

B

HRP

SA

AP

AP AP

AP

AP

1˚ Ab

2˚ Ab

T

B

T

B

SA

SASA

T

B

T

B

SA

SA

HRP

SA

B

T

B

T

T

B

B

T

SA

BAP

AP

AP

SA

1˚ Ab

2˚ Ab

T

B

T

B

SA

SASA

T

B

T

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SA

SA

HRP

SA

SA

B

T

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T

T

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B

T

SA

B

F

F

F

F

F

F F

F

SA

SA

Chromogenic with SA-HRP

Chromogenic with SA-AP

Fluorescence with SA-Fluorophore

NOTE: TSA and Avidin Biotin Complex (ABC) —TSA can be integrated into systems currently using the ABC method, either before or after the ABC step.



Amplification and Visualization Procedures

After signal amplification, the depositedbiotin can be directly visualized by any of the following methods:

Addition of SA-HRP followed by horseradishperoxidase catalyzed chromogen

Addition of SA-AP followed by alkalinephosphatase catalyzed chromogen

Addition of SA-Fluorophore(SA-Fluorescein)(SA-Coumarin)(SA-Texas Red®)

viewed by fluorescence microscopy

APAP

APAP

APAP

HRP

Ab Ab

HRPHRP

HRP

SA

HRP

SA

HR

P

SA

HR

P

SAHRP

SA

HRP

SA

HRP

SAT

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T

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SA

SASA SA SA

SA

HRP

SAT

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SA SA SA

SA

HRP

SAT

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F

F F

FF

F

SA

B F D

Chromogenic with SA-AP

Fluorescence with SA-Fluorophore

Probe Labeling Option

Chromogenic with SA-HRP

Detection

First, consider your research requirements:

• Select chromogenic or fluorescence detection based on your laboratory equipment and your need for a permanent record.

• Eliminate biotin interference by considering the level of endogenous biotin in your specimens and your desire to reduce background or eliminate biotin-quenching procedures.

• Choose from several fluorophores based on your preferred fluors and your interest in performing multi-target detection.

• Determine the degree of amplification you need, taking into account the abundance of the protein or gene in your specimen and your need to conserve antibody or probe.

Then, based on your experience, choose one of three TSA formats:

• Complete TSA Systems: complete tyramide signal amplification kits include labeled tyramide, amplificationdiluent, streptavidin HRP, blocking reagent and comprehensive instruction manuals. Additional antibodies,conjugates, and chromogens may be required for your optimal protocol and may be purchased separately.

• Complete TSA Plus Systems: tyramide signal amplification kits that provide 10 to 20 times the sensitivity enhancement of regular TSA systems.

• TSA Reagent Packs: stand-alone amplification reagents that provide researchers experienced with TSA the flexibility to develop their own protocols.

There’s a TSA systemPerkinElmer offers a variety of TSA systems to enable every lab to benefit from

the sensitivity enhancements of this outstanding technology. You’ll maximize the

difference that TSA makes in your IHC and ISH applications when you select a TSA

system based on your research requirements and the experience of your lab.

Mouse Brain, 20x magnification, 15 second exposure

Conventional detection with fluorescein conjugated 2° antibody. Dilution 1:100

Standard TSA fluorescein. Dilution 1:10,000

TSA Plus fluorescein. Dilution 1:1,000,000

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for your laboratory…


Embryonic mousebrain. Histone H4mRNA ISH, usingTSA Plus Cyanine 3and MAP2immunoreactivity(IHC) with TSA PlusFluorescein System.

Multi-target detection in the same sample.Detection of Arg-Vasopressin and Oxytocinin rat brain using TSACyanine 3 and TSACyanine 5, respectively.

Complete TSA Systems

TSA Fluorescence Systems

• Used for direct fluorescence detection in IHC and ISH applications.

• While not as sensitive as TSA Plus systems, allow significant reductions in antibody or probe with necessary sensitivity in many applications.

TSA Biotin Systems

• Designed for chromogenic detection in ISH and IHC applications.

• Can also be used for indirect fluorescence detection to maximize fluorescent signal amplification.

• Use streptavidin-conjugated chromogens or fluorophores for visualization.

Complete TSA Plus Systems

TSA Plus Fluorescence Systems

• Used for direct fluorescence detection in IHC and ISH applications.

• Provide very high sensitivity in ISH compared to traditional methods.

• While developed specifically for ISH applications,these systems can also be used with IHC applications.

• Choose from five different fluorphores: fluorescein,TMR, coumarin, Cyanine-3, or Cyanine-5.

TSA Plus Fluorescence Palette Systems

• Convenient and cost-effective way of combining two fluors for multi-target detection procedures.

• Choose from four different two-fluor combinations.

• Evaluation of TSA fluorescence detection or exploringmulti-target detection is easy with our TSA PlusFluorescence Palette System, a trial-sized kit withfour different fluors packaged together. Each fluor has sufficient reagent for 10–35 slides.

IHC detection of CD3 antigen in serial sections of paraffin-embedded human tonsil. (A) Rabbit anti-CD3 1:400, biotinylatedanti-rabbit, ABC, with DAB chromogenic detection. (B) TSABiotin: Rabbit anti-CD3 1:400, biotinylated anti-rabbit, SA-HRP,biotinyl tyramide, ABC, with DAB chromogenic detection.(Courtesy of F. van den Berg and A. de Koning, Dept. Pathology,AMC, Amsterdam, The Netherlands.)

A

B

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…with the flexibility to match your research and application needs.TSA Plus DNP Systems

• Designed for biotin-free chromogenic detection in IHC and ISH applications.

• Use to reduce background associated with endogenous biotin.

• Simple protocol eliminates biotin-quenching procedures.

• The ISH protocol included is specifically optimizedfor using with popular DIG-labeled probes, although any hapten-labeled probe can be used.

• Enhances sensitivity of existing DIG labeling protocols.

TSA Reagent Packs

TSA Reagent Packs are stand-alone tyramide (SAT)kits for signal amplification that provide flexibility to researchers for TSA protocol development.Researchers must supply their own blocking buffer,HRP and other reagents as needed. If you have notused TSA Signal Amplification reagents before, westrongly suggest purchasing one of our complete TSAsystems, which include a set of quality controlledreagents optimized to work well together.

Use versatile, flexible TSA to enhancesensitivity in many applications

TSA makes a difference in the sensitivity of anyapplication using an antibody or gene probe thatallows the addition of HRP into its protocol:

• ELISA: amplify signal in microplate-based assays,greatly increasing sensitivity or reducing the use ofantibodies. Use our convenient ELAST® ELISAAmplification System.

• Protein microarrays: detect low abundance proteinsin differential protein expression experiments. Useour TSA AUTOMATED Kits with our ProteinArray™

Workstation, a highly flexible, fully automated sys-tem for processing multiple protein microarrays.

• Genomic microarrays: apply TSA to your cDNAdifferential gene expression studies using ourMICROMAX™ TSA Labeling and Detection Kits.

TSA is compatible with all commondetection methods:

• EnzymaticABC (avidin-biotinylated HRP complex)

PAP (peroxidase anti-peroxidase)

APAP (alkaline phosphatase anti-alkaline phosphatase)

• ChromogenicBCIP/NBT (using alkaline phosphatase-conjugated secondary antibody or avidin)

DAB (using second application of HRP-conjugated secondary antibody or avidin)

• Electron Microscopy (EM)Gold conjugate

Immunogold detection systems

• Fluorescence (Direct and Indirect)

Quick GlanceExpert technical support with one phone call

PerkinElmer Life and Analytical Sciences gives you easy access to a worldwide network of localsupport offices, staffed by highly trained professionals who speak your language. You’ll get expertassistance for all of your questions — no matter where you are located — when you add TSA toyour protocols. Contact us at (800) 762-4000 or (+1) 203-925-4602, or by e-mail [email protected] or [email protected].

www.perkinelmer.com

TSA Fluorescence Systems

Product No. of Slides Product No.

TSA Fluorescein System 50–150 NEL701A001KT100–300 NEL701001KT

TSA TMR System 100–300 NEL702001001KTTSA Coumarin System 100–300 NEL703001KTTSA Cyanine 3 System 50–150 NEL704A001KTTSA Cyanine 5 System* 50–150 NEL705A001KT

TSA Biotin Systems


TSA Biotin System 50–150 NEL700A001KT200–600 NEL700001KT

TSA Plus Fluorescence Systems


TSA Plus Fluorescein System 50–150 NEL741001KT250–750 NEL741B001KT

TSA Plus TMR System 50–150 NEL742001KT250–750 NEL742B001KT

TSA Plus Cyanine 3 System 50–150 NEL744001KT250–750 NEL744B001KT

TSA Plus Cyanine 5 System* 50–150 NEL745001KT250–750 NEL745B001KT

TSA Plus Fluorescence Palette Systems


TSA Plus Cyanine 3/Cyanine 5 System* 50–150 NEL752001KTTSA Plus Cyanine 3/Fluorescein System 50–150 NEL753001KTTSA Plus Cyanine 5/Fluorescein System* 50–150 NEL754001KTTSA Plus Fluorescein/TMR System 50–150 NEL756001KTTSA Plus Fluorescence Palette System (contains one each of Fluorescein, TMR, Cyanine 3 and Cyanine 5)* 10–35 NEL760001KT

TSA Plus DNP Systems (Biotin-free)


TSA Plus DNP (AP) System 25–75 NEL746B001KT50–150 NEL746A001KT

TSA Plus DNP (HRP) System 25–75 NEL747B001KT50–150 NEL747A001KT

TSA Reagent Packs


TSA Biotin Tyramide Reagent Pack 200–600 SAT700001EA1,000–3,000 SAT700B001EA

TSA Fluorescein Tyramide Reagent Pack 100–300 SAT701001EA500–1,500 SAT701B001EA

TSA TMR Tyramide Reagent Pack 100–300 SAT702001EATSA Cyanine 3 Tyramide Reagent Pack 50–150 SAT704A001EA

250–750 SAT704B001EATSA Cyanine 5 Tyramide Reagent Pack* 50–150 SAT705A001EA

*Cyanine 5 tyramides cannot be visualized with most digital cameras.

Ordering Information

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For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices

©2007 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. TSA, ProteinArray, MICROMAX and ELAST aretrademarks or registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. Texas Red is a registered trademark of Teletype Corp.All other trademarks not owned by PerkinElmer, Inc. that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors.

007703_01 Printed in USA

PerkinElmer Life and Analytical Sciences710 Bridgeport AvenueShelton, CT 06484-4794 USAPhone: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com

Complementary Products for Use with TSAThese high quality conjugates, chromogens and secondary antibodies can be used with TSA Assay Systems and Reagent Packs.

Chromogens & Conjugates

Product Product No.

BCIP/NBT (AP substrate) NEL937001PKDAB (HRP substrate) NEL938001EAAntifluorescein-AP NEF709001PKAntifluorescein-HRP NEF710001EAStreptavidin Fluorescein NEL720001EAStreptavidin Texas Red® NEL721001EAStreptavidin Coumarin NEL722001EAStreptavidin-HRP NEL750001EAStreptavidin-AP NEL751001EA

Secondary Antibodies

Antibody Label Size Concentration Product No.

Anti-human IgG (goat)* AP 1 mL 1 mg/mL NEF801001EAAnti-human IgG (goat)* HRP 1 mL 1 mg/mL NEF802001EAAnti-human IgG (goat) Biotin 0.5 mg Lyophilized NEF803001EAAnti-mouse IgG (goat) AP 1 mL 1 mg/mL NEF821001EAAnti-mouse IgG (goat) HRP 1 mL 1 mg/mL NEF822001EAAnti-mouse IgG (goat) Biotin 0.5 mg Lyophilized NEF823001EAAnti-rabbit IgG (goat) AP 1 mL 1 mg/mL NEF811001EAAnti-rabbit IgG (goat) HRP 1 mL 1 mg/mL NEF812001EAAnti-rabbit IgG (goat) Biotin 0.5 mg Lyophilized NEF813001EA

*Bovine serum albumin added as a stabilizer.

TSA technology from PerkinElmer—the difference is clear!

Add TSA to your protocols and you, too, will see the difference. Learn more about the exciting enhancements you’ll see when you use the ultimate technology for maximizing detection sensitivity. For more information, visit www.perkinelmer.com/tsa, phone (800) 762-4000 or (+1) 203-925-4602, or contact your local sales representative. For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices.