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TSA Signal Amplification (TSA) SystemsSee the difference with TSA—
the ultimate technology for unparalleled detection
sensitivity
TSA Signal Amplification (TSA) Systems
2
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 both chromogenic and fluorescent signals in many applications. TSA can be used in any application that allows the addition 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
Quick Glance
TSA is easily integrated into any protocol after an initial addition of horseradish peroxidase (HRP). The HRP is used to catalyze the deposition and binding of a labeled (e.g., biotin, DNP or other labeling moieties) tyramide onto tissue sections or cell preparations previously blocked with proteins. This reaction is quick (less than 10 minutes). The binding is covalent because the reaction intermediately dimerizes with tyrosine residues 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
4
Increase Sensitivity
Maximizing the sensitivity of methods aimed at the cellular localization of proteins and nucleic acids is of particular importance when target levels are known or suspected to be low. Addition of TSA to your protocol may reveal localized targets previously undetectable and therefore unsuspected — without loss of resolution or increase in background.
See the difference in your
Do Multi-Target Detection
TSA systems have been designed to allow utilization of various chromogens or fluorophores. This flexibility facilitates applications in which multi-target detection in 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 chromosome 15, band D2, mouse metaphase chromosomes. Sensitivity improvement with TSA allows use of smaller (cDNA, oligos) probes for more precise localization. Detection of single copy genomic sequence was achieved using a probe less than 2 kb.
Conserve Antibody or Probe
Application of TSA may be beneficial even when an increase in sensitivity is not required because TSA allows use of less antibody or probe. Typically, the same 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 TSA enhancement, (B) Anti-EBV dilution 1:25,000, enhanced by TSA. (Courtesy of R. Von Wasielewski and S. Gignac, Pathologisches Institut de Medizinischen Hochscule, Hannover, Germany.)
A
B
A
B
CHR 15 D2
Eliminate PCR
TSA is simpler and faster than IS-PCR with equivalent sensitivity. 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.)
www.perkinelmer.com 5
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 images than other methods.
Comparison of direct immunofluorescent staining and TSA- enhanced direct fluorescent staining of CMV infected cells. (A) Direct fluorescent staining. (B) Direct fluorescent staining enhanced by TSA.
Reduce Interference
Biotin-free TSA Plus DNP Systems provide a simple protocol and eliminate biotin quenching procedures. Biotin-free formats also eliminate background problems.
Detection of p53 mRNA in lung tissue. Digoxigenin-labeled p53 RNA 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 for radiometric detection, and achieve equivalent sensitivity and resolution.
Comparison of radiometric and TSA-amplified ISH detection of muscarinic receptor mRNA in rat brain hippocampus. M-1 oligo-nucleotide probes were labeled with 33P and fluorescein- N6-ddATP by 3' end-labeling and were hybridized to frozen rat brain tissue sections. (A) Autoradiography of 33P was carried out for one month. (B) TSA amplification with DAB.
A
B
A
B
A
B
6
Immunohistochemistry
Blocking Step 30 min
Incubation of fluorophore tyramide 3–10 min
Wash Step 3 x 5 min
Production of Reactive Tyramide by HRP
Key:
B = Biotin F = Fluorophore T = Tyramide SA = Streptavidin HRP = Horseradish Peroxidase AP = Alkaline Phosphatase
TSA Plus Cyanine 3. Dilution 1:10,000,000
Conventional detection with Cyanine 3 conjugated 2 antibody. Dilution 1:1,000
Standard TSA Cyanine 3. Dilution 1:100,000
Mouse Brain, 20x magnification, 2 second exposure
www.perkinelmer.com 7
Key:
B = Biotin F = Fluorophore T = Tyramide SA = Streptavidin HRP = Horseradish Peroxidase AP = Alkaline Phosphatase D = Digoxigenin
Amplification Actual Time and Visualization Added to Procedures Protocol
Blocking Step 30 min
Incubation of fluorophore tyramide 3–10 min
Wash Step 3 x 5 min
Production of Reactive Tyramide by HRP
Embryonic mouse brain histone H4 mRNA ISH, using TSA Plus Cyanine 3 and MAP2 immunoreactivity (IHC) with TSA Plus Fluorescein System.
on
8
(Biotin-based or Biotin-free) Immunohistochemistry
Blocking Step 30 min
Incubation of biotinyl 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
Detection of Target
Detection of Target Amplification Actual Time and Visualization Added to Procedures Protocol
Blocking Step 30 min
Incubation of biotinyl 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
Production of Reactive Tyramide by HRP
rescence Detection
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 catalyzed chromogen
Addition of SA-AP followed by alkaline phosphatase catalyzed chromogen
Addition of SA-Fluorophore (SA-Fluorescein) (SA-Coumarin) (SA-Texas Red®)
viewed by fluorescence microscopy
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.
www.perkinelmer.com 11
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 catalyzed chromogen
Addition of SA-AP followed by alkaline phosphatase catalyzed chromogen
Addition of SA-Fluorophore (SA-Fluorescein) (SA-Coumarin) (SA-Texas Red®)
viewed by fluorescence microscopy
• 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, amplification diluent, 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 system PerkinElmer 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
12
www.perkinelmer.com 13
Embryonic mouse brain. Histone H4 mRNA ISH, using TSA Plus Cyanine 3 and MAP2 immunoreactivity (IHC) with TSA Plus Fluorescein System.
Multi-target detection in the same sample. Detection of Arg- Vasopressin and Oxytocin in rat brain using TSA Cyanine 3 and TSA Cyanine 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 exploring multi-target detection is easy with our TSA Plus Fluorescence Palette System, a trial-sized kit with four 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, biotinylated anti-rabbit, ABC, with DAB chromogenic detection. (B) TSA Biotin: 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
14
…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 optimized for 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 not used TSA Signal Amplification reagents before, we strongly suggest purchasing one of our complete TSA systems, which include a set of quality controlled reagents optimized to work well together.
Use versatile, flexible TSA to enhance sensitivity in many applications
TSA makes a difference in the sensitivity of any application using an antibody or gene probe that allows the addition of HRP into its protocol:
• ELISA: amplify signal in microplate-based assays, greatly increasing sensitivity or reducing the use of antibodies. Use our convenient ELAST® ELISA Amplification System.
• Protein microarrays: detect low abundance proteins in differential protein expression experiments. Use our 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 cDNA differential gene expression studies using our MICROMAX™ TSA Labeling and Detection Kits.
TSA is compatible with all common detection methods:
• Enzymatic ABC (avidin-biotinylated HRP complex)
PAP (peroxidase anti-peroxidase)
• Chromogenic BCIP/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
Quick Glance Expert technical support with one phone call
PerkinElmer Life and Analytical Sciences gives you easy access to a worldwide network of local support offices, staffed by highly trained professionals who speak your language. You’ll get expert assistance for all of your questions — no matter where you are located — when you add TSA to your protocols. Contact us at (800) 762-4000 or (+1) 203-925-4602, or by e-mail at te[email protected] or [email protected].
www.perkinelmer.com
TSA Fluorescein System 50–150 NEL701A001KT 100–300 NEL701001KT
TSA TMR System 100–300 NEL702001001KT TSA Coumarin System 100–300 NEL703001KT TSA Cyanine 3 System 50–150 NEL704A001KT TSA Cyanine 5 System* 50–150 NEL705A001KT
TSA Biotin Systems
TSA Biotin System 50–150 NEL700A001KT 200–600 NEL700001KT
TSA Plus Fluorescence Systems
TSA Plus Fluorescein System 50–150 NEL741001KT 250–750 NEL741B001KT
TSA Plus TMR System 50–150 NEL742001KT 250–750 NEL742B001KT
TSA Plus Cyanine 3 System 50–150 NEL744001KT 250–750 NEL744B001KT
TSA Plus Cyanine 5 System* 50–150 NEL745001KT 250–750 NEL745B001KT
TSA Plus Fluorescence Palette Systems
Product No. of Slides Product No.
TSA Plus Cyanine 3/Cyanine 5 System* 50–150 NEL752001KT TSA Plus Cyanine 3/Fluorescein System 50–150 NEL753001KT TSA Plus Cyanine 5/Fluorescein System* 50–150 NEL754001KT TSA Plus Fluorescein/TMR System 50–150 NEL756001KT TSA Plus Fluorescence Palette System (contains one each of Fluorescein, TMR, Cyanine 3 and Cyanine 5)* 10–35 NEL760001KT
TSA Plus DNP Systems (Biotin-free)
Product No. of Slides Product No.
TSA Plus DNP (AP) System 25–75 NEL746B001KT 50–150 NEL746A001KT
TSA Plus DNP (HRP) System 25–75 NEL747B001KT 50–150 NEL747A001KT
TSA Reagent Packs
Product No. of Slides Product No.
TSA Biotin Tyramide Reagent Pack 200–600 SAT700001EA 1,000–3,000 SAT700B001EA
TSA Fluorescein Tyramide Reagent Pack 100–300 SAT701001EA 500–1,500 SAT701B001EA
TSA TMR Tyramide Reagent Pack 100–300 SAT702001EA TSA Cyanine 3 Tyramide Reagent Pack 50–150 SAT704A001EA
250–750 SAT704B001EA TSA Cyanine 5 Tyramide Reagent Pack* 50–150 SAT705A001EA
*Cyanine 5 tyramides cannot be visualized with most digital cameras.
Ordering Information
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 are trademarks 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 Sciences 710 Bridgeport Avenue Shelton, CT 06484-4794 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com
Complementary Products for Use with TSA These high quality conjugates, chromogens and secondary antibodies can be used with TSA Assay Systems and Reagent Packs.
Chromogens & Conjugates
Secondary Antibodies
Antibody Label Size Concentration Product No.
Anti-human IgG (goat)* AP 1 mL 1 mg/mL NEF801001EA Anti-human IgG (goat)* HRP 1 mL 1 mg/mL NEF802001EA Anti-human IgG (goat) Biotin 0.5 mg Lyophilized NEF803001EA Anti-mouse IgG (goat) AP 1 mL 1 mg/mL NEF821001EA Anti-mouse IgG (goat) HRP 1 mL 1 mg/mL NEF822001EA Anti-mouse IgG (goat) Biotin 0.5 mg Lyophilized NEF823001EA Anti-rabbit IgG (goat) AP 1 mL 1 mg/mL NEF811001EA Anti-rabbit IgG (goat) HRP 1 mL 1 mg/mL NEF812001EA Anti-rabbit IgG (goat) Biotin 0.5 mg Lyophilized NEF813001EA
*Bovine serum albumin added as a stabilizer.
TSA technology from PerkinElmer— the difference is clear!
TSA Signal Amplification (TSA) Systems
2
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 both chromogenic and fluorescent signals in many applications. TSA can be used in any application that allows the addition 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
Quick Glance
TSA is easily integrated into any protocol after an initial addition of horseradish peroxidase (HRP). The HRP is used to catalyze the deposition and binding of a labeled (e.g., biotin, DNP or other labeling moieties) tyramide onto tissue sections or cell preparations previously blocked with proteins. This reaction is quick (less than 10 minutes). The binding is covalent because the reaction intermediately dimerizes with tyrosine residues 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
4
Increase Sensitivity
Maximizing the sensitivity of methods aimed at the cellular localization of proteins and nucleic acids is of particular importance when target levels are known or suspected to be low. Addition of TSA to your protocol may reveal localized targets previously undetectable and therefore unsuspected — without loss of resolution or increase in background.
See the difference in your
Do Multi-Target Detection
TSA systems have been designed to allow utilization of various chromogens or fluorophores. This flexibility facilitates applications in which multi-target detection in 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 chromosome 15, band D2, mouse metaphase chromosomes. Sensitivity improvement with TSA allows use of smaller (cDNA, oligos) probes for more precise localization. Detection of single copy genomic sequence was achieved using a probe less than 2 kb.
Conserve Antibody or Probe
Application of TSA may be beneficial even when an increase in sensitivity is not required because TSA allows use of less antibody or probe. Typically, the same 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 TSA enhancement, (B) Anti-EBV dilution 1:25,000, enhanced by TSA. (Courtesy of R. Von Wasielewski and S. Gignac, Pathologisches Institut de Medizinischen Hochscule, Hannover, Germany.)
A
B
A
B
CHR 15 D2
Eliminate PCR
TSA is simpler and faster than IS-PCR with equivalent sensitivity. 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.)
www.perkinelmer.com 5
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 images than other methods.
Comparison of direct immunofluorescent staining and TSA- enhanced direct fluorescent staining of CMV infected cells. (A) Direct fluorescent staining. (B) Direct fluorescent staining enhanced by TSA.
Reduce Interference
Biotin-free TSA Plus DNP Systems provide a simple protocol and eliminate biotin quenching procedures. Biotin-free formats also eliminate background problems.
Detection of p53 mRNA in lung tissue. Digoxigenin-labeled p53 RNA 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 for radiometric detection, and achieve equivalent sensitivity and resolution.
Comparison of radiometric and TSA-amplified ISH detection of muscarinic receptor mRNA in rat brain hippocampus. M-1 oligo-nucleotide probes were labeled with 33P and fluorescein- N6-ddATP by 3' end-labeling and were hybridized to frozen rat brain tissue sections. (A) Autoradiography of 33P was carried out for one month. (B) TSA amplification with DAB.
A
B
A
B
A
B
6
Immunohistochemistry
Blocking Step 30 min
Incubation of fluorophore tyramide 3–10 min
Wash Step 3 x 5 min
Production of Reactive Tyramide by HRP
Key:
B = Biotin F = Fluorophore T = Tyramide SA = Streptavidin HRP = Horseradish Peroxidase AP = Alkaline Phosphatase
TSA Plus Cyanine 3. Dilution 1:10,000,000
Conventional detection with Cyanine 3 conjugated 2 antibody. Dilution 1:1,000
Standard TSA Cyanine 3. Dilution 1:100,000
Mouse Brain, 20x magnification, 2 second exposure
www.perkinelmer.com 7
Key:
B = Biotin F = Fluorophore T = Tyramide SA = Streptavidin HRP = Horseradish Peroxidase AP = Alkaline Phosphatase D = Digoxigenin
Amplification Actual Time and Visualization Added to Procedures Protocol
Blocking Step 30 min
Incubation of fluorophore tyramide 3–10 min
Wash Step 3 x 5 min
Production of Reactive Tyramide by HRP
Embryonic mouse brain histone H4 mRNA ISH, using TSA Plus Cyanine 3 and MAP2 immunoreactivity (IHC) with TSA Plus Fluorescein System.
on
8
(Biotin-based or Biotin-free) Immunohistochemistry
Blocking Step 30 min
Incubation of biotinyl 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
Detection of Target
Detection of Target Amplification Actual Time and Visualization Added to Procedures Protocol
Blocking Step 30 min
Incubation of biotinyl 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
Production of Reactive Tyramide by HRP
rescence Detection
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 catalyzed chromogen
Addition of SA-AP followed by alkaline phosphatase catalyzed chromogen
Addition of SA-Fluorophore (SA-Fluorescein) (SA-Coumarin) (SA-Texas Red®)
viewed by fluorescence microscopy
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.
www.perkinelmer.com 11
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 catalyzed chromogen
Addition of SA-AP followed by alkaline phosphatase catalyzed chromogen
Addition of SA-Fluorophore (SA-Fluorescein) (SA-Coumarin) (SA-Texas Red®)
viewed by fluorescence microscopy
• 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, amplification diluent, 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 system PerkinElmer 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
12
www.perkinelmer.com 13
Embryonic mouse brain. Histone H4 mRNA ISH, using TSA Plus Cyanine 3 and MAP2 immunoreactivity (IHC) with TSA Plus Fluorescein System.
Multi-target detection in the same sample. Detection of Arg- Vasopressin and Oxytocin in rat brain using TSA Cyanine 3 and TSA Cyanine 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 exploring multi-target detection is easy with our TSA Plus Fluorescence Palette System, a trial-sized kit with four 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, biotinylated anti-rabbit, ABC, with DAB chromogenic detection. (B) TSA Biotin: 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
14
…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 optimized for 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 not used TSA Signal Amplification reagents before, we strongly suggest purchasing one of our complete TSA systems, which include a set of quality controlled reagents optimized to work well together.
Use versatile, flexible TSA to enhance sensitivity in many applications
TSA makes a difference in the sensitivity of any application using an antibody or gene probe that allows the addition of HRP into its protocol:
• ELISA: amplify signal in microplate-based assays, greatly increasing sensitivity or reducing the use of antibodies. Use our convenient ELAST® ELISA Amplification System.
• Protein microarrays: detect low abundance proteins in differential protein expression experiments. Use our 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 cDNA differential gene expression studies using our MICROMAX™ TSA Labeling and Detection Kits.
TSA is compatible with all common detection methods:
• Enzymatic ABC (avidin-biotinylated HRP complex)
PAP (peroxidase anti-peroxidase)
• Chromogenic BCIP/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
Quick Glance Expert technical support with one phone call
PerkinElmer Life and Analytical Sciences gives you easy access to a worldwide network of local support offices, staffed by highly trained professionals who speak your language. You’ll get expert assistance for all of your questions — no matter where you are located — when you add TSA to your protocols. Contact us at (800) 762-4000 or (+1) 203-925-4602, or by e-mail at te[email protected] or [email protected].
www.perkinelmer.com
TSA Fluorescein System 50–150 NEL701A001KT 100–300 NEL701001KT
TSA TMR System 100–300 NEL702001001KT TSA Coumarin System 100–300 NEL703001KT TSA Cyanine 3 System 50–150 NEL704A001KT TSA Cyanine 5 System* 50–150 NEL705A001KT
TSA Biotin Systems
TSA Biotin System 50–150 NEL700A001KT 200–600 NEL700001KT
TSA Plus Fluorescence Systems
TSA Plus Fluorescein System 50–150 NEL741001KT 250–750 NEL741B001KT
TSA Plus TMR System 50–150 NEL742001KT 250–750 NEL742B001KT
TSA Plus Cyanine 3 System 50–150 NEL744001KT 250–750 NEL744B001KT
TSA Plus Cyanine 5 System* 50–150 NEL745001KT 250–750 NEL745B001KT
TSA Plus Fluorescence Palette Systems
Product No. of Slides Product No.
TSA Plus Cyanine 3/Cyanine 5 System* 50–150 NEL752001KT TSA Plus Cyanine 3/Fluorescein System 50–150 NEL753001KT TSA Plus Cyanine 5/Fluorescein System* 50–150 NEL754001KT TSA Plus Fluorescein/TMR System 50–150 NEL756001KT TSA Plus Fluorescence Palette System (contains one each of Fluorescein, TMR, Cyanine 3 and Cyanine 5)* 10–35 NEL760001KT
TSA Plus DNP Systems (Biotin-free)
Product No. of Slides Product No.
TSA Plus DNP (AP) System 25–75 NEL746B001KT 50–150 NEL746A001KT
TSA Plus DNP (HRP) System 25–75 NEL747B001KT 50–150 NEL747A001KT
TSA Reagent Packs
Product No. of Slides Product No.
TSA Biotin Tyramide Reagent Pack 200–600 SAT700001EA 1,000–3,000 SAT700B001EA
TSA Fluorescein Tyramide Reagent Pack 100–300 SAT701001EA 500–1,500 SAT701B001EA
TSA TMR Tyramide Reagent Pack 100–300 SAT702001EA TSA Cyanine 3 Tyramide Reagent Pack 50–150 SAT704A001EA
250–750 SAT704B001EA TSA Cyanine 5 Tyramide Reagent Pack* 50–150 SAT705A001EA
*Cyanine 5 tyramides cannot be visualized with most digital cameras.
Ordering Information
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 are trademarks 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 Sciences 710 Bridgeport Avenue Shelton, CT 06484-4794 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com
Complementary Products for Use with TSA These high quality conjugates, chromogens and secondary antibodies can be used with TSA Assay Systems and Reagent Packs.
Chromogens & Conjugates
Secondary Antibodies
Antibody Label Size Concentration Product No.
Anti-human IgG (goat)* AP 1 mL 1 mg/mL NEF801001EA Anti-human IgG (goat)* HRP 1 mL 1 mg/mL NEF802001EA Anti-human IgG (goat) Biotin 0.5 mg Lyophilized NEF803001EA Anti-mouse IgG (goat) AP 1 mL 1 mg/mL NEF821001EA Anti-mouse IgG (goat) HRP 1 mL 1 mg/mL NEF822001EA Anti-mouse IgG (goat) Biotin 0.5 mg Lyophilized NEF823001EA Anti-rabbit IgG (goat) AP 1 mL 1 mg/mL NEF811001EA Anti-rabbit IgG (goat) HRP 1 mL 1 mg/mL NEF812001EA Anti-rabbit IgG (goat) Biotin 0.5 mg Lyophilized NEF813001EA
*Bovine serum albumin added as a stabilizer.
TSA technology from PerkinElmer— the difference is clear!
