TSA Signal Amplification (TSA) Systems. See the difference with TSA the ultimate technology for unparalleled detection sensitivity

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1 Signal Amplification () Systems See the difference with the ultimate technology for unparalleled detection sensitivity

2 or unparalled sensitivity, technology Increase your immunohistochemistry and in situ hybridization detection capabilities with PerkinElmer s proprietary yramide Signal Amplification technology ( ) and see clearly why is the standard for superior IHC and ISH sensitivity in labs around the world. he remarkable sensitivity enhancements that 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 technology to a wide range of scientific research fields. Add to your protocols and you, too, will see the difference. yramide Signal Amplification is a patented* technology invented by PerkinElmer scientists that amplifies both chromogenic and fluorescent signals in many applications. can be used in any application that allows the addition of horseradish peroxidase () into its protocol, such as immunohistochemistry, in situ hybridization, ELI and microarray-based differential gene and protein expression studies. In standard IHC and ISH protocols, use of results in a significant increase in sensitivity, without loss of resolution or increase in background. * is protected by US patents: 5,731,158, 5,583,001, and 5,196,306 and foreign equivalents. PerkinElmer is the world s sole source of patented tech 2

3 makes all the difference Quick Glance Innovative amplification technology maintains signal clarity is easily integrated into any protocol after an initial addition of horseradish peroxidase (). he 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. his reaction is quick (less than 10 minutes). he binding is covalent because the reaction intermediately dimerizes with tyrosine residues on the surface-bound endogenous proteins. hese labels can then be detected by standard chromogenic or fluorescent techniques. Since the added labels are only deposited proximal to the enzyme site, there is minimal, if any, loss of resolution. Diagram of direct fluorescence detection. nology the standard for superior IHC and ISH sensitivity. 3

4 See the difference in your can provide numerous benefits when you add it to your immunohistochemistry or 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 to your protocol may reveal localized targets previously undetectable and therefore unsuspected without loss of resolution or increase in background. Conserve Antibody or Probe Application of may be beneficial even when an increase in sensitivity is not required because allows use of less antibody or probe. ypically, 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. CHR 15 D2 Gene mapping of c-myc, exon 2 gene (855 bp) mapped to chromosome 15, band D2, mouse metaphase chromosomes. Sensitivity improvement with 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. A IHC of EV antigen in Hodgkin s Lymphoma of mixed cellularity. Comparison shows (A) Anti-EV dilution 1:25, without enhancement, () Anti-EV dilution 1:25,000, enhanced by. (Courtesy of R. Von Wasielewski and S. Gignac, Pathologisches Institut de Medizinischen Hochscule, Hannover, Germany.) Do Multi-arget Detection systems have been designed to allow utilization of various chromogens or fluorophores. his flexibility facilitates applications in which multi-target detection in the same sample is of interest. Eliminate PCR is simpler and faster than IS-PCR with equivalent sensitivity. It detects even low or single copy genes. No special equipment is required. Multi-target detection enables simultaneous visualization of multiple targets. A Detection of HIV-1 DNA in formaldehyde-fixed lymph node tissues from positive autopsy material. (A) In situ PCR, 20 cycles. () -enhanced ISH. (Courtesy of Dr. J. Luka and C. Afflerbach, Eastern Virginia Medical School, Norfolk, VA.) 4

5 research with in your protocol in situ hybridization protocols. Use of is particularly valuable when you need to: Obtain High Resolution provides exquisite resolution for clearer images than other methods. Get aster Results See results within a day versus up to a week for radiometric detection, and achieve equivalent sensitivity and resolution. A Comparison of direct immunofluorescent staining and enhanced direct fluorescent staining of CMV infected cells. (A) Direct fluorescent staining. () Direct fluorescent staining enhanced by. A Reduce Interference iotin-free Plus DNP Systems provide a simple protocol and eliminate biotin quenching procedures. iotin-free formats also eliminate background problems. Comparison of radiometric and -amplified ISH detection of muscarinic receptor mrna in rat brain hippocampus. M-1 oligo-nucleotide probes were labeled with 33 P and fluorescein- N 6 -dd by 3' end-labeling and were hybridized to frozen rat brain tissue sections. (A) Autoradiography of 33 P was carried out for one month. () amplification with DA. A 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/cip-n (60 minute substrate incubation), and () Plus DNP with alkaline phosphatase/cip-n (15 minute substrate incubation). 5

6 Direct luorescence Detecti Immunohistochemistry Detection of arget 1 Ab Key: = iotin = luorophore = yramide = Streptavidin = Horseradish Peroxidase = Alkaline Phosphatase Amplification and Visualization Procedures locking Step Actual ime Added to Protocol 30 min LOCKING REAGEN ARGE Signal Amplification Production of Reactive yramide by Incubation of fluorophore tyramide 3 10 min 1 Ab Visualization via luorescence Microscopy Mouse rain, 20x magnification, 2 second exposure Conventional detection with Cyanine 3 conjugated 2 antibody. Dilution 1:1,000 Standard Cyanine 3. Dilution 1:100,000 Plus Cyanine 3. Dilution 1:10,000,000 6

7 on In Situ Hybridization Key: Detection of arget = iotin = luorophore = yramide = Streptavidin = Horseradish Peroxidase = Alkaline Phosphatase D = Digoxigenin Amplification and Visualization Procedures Actual ime Added to Protocol ARGE PROE LOCKING REAGEN locking Step 30 min Signal Amplification Production of Reactive yramide by Incubation of fluorophore tyramide 3 10 min Visualization via luorescence Microscopy Embryonic mouse brain histone H4 mrna ISH, using Plus Cyanine 3 and M2 immunoreactivity (IHC) with Plus luorescein System. 7

8 Indirect Chromogenic or luo Immunohistochemistry (iotin-based or iotin-free) Detection of arget Amplification and Visualization Procedures Actual ime Added to Protocol 1 Ab locking Step 30 min LOCKING REAGEN Signal Amplification ARGE Production of Reactive yramide by Incubation of biotinyl tyramide 3 10 min 1 Ab Detection of Amplified Signal Addition of enzyme conjugate 30 min 1 Ab Visualization Incubation of chromogen or fluorophore 1 Ab 8

9 rescence Detection In Situ Hybridization (iotin-based or iotin-free) Detection of arget ARGE PROE LOCKING REAGEN Amplification and Visualization Procedures locking Step Actual ime Added to Protocol 30 min Signal Amplification Production of Reactive yramide by Incubation of biotinyl tyramide 3 10 min Addition of enzyme conjugate 30 min Detection of Amplified Signal Incubation of chromogen or fluorophore Visualization 9

10 Visualization Options for Indirect Immunohistochemistry Chromogenic with - Amplification and Visualization Procedures 1 Ab After signal amplification, the deposited biotin can be directly visualized by any of the following methods: Addition of - followed by horseradish peroxidase catalyzed chromogen Chromogenic with - Addition of - followed by alkaline phosphatase catalyzed chromogen luorescence with -luorophore 1 Ab Addition of -luorophore (-luorescein) (-Coumarin) (-exas Red ) viewed by fluorescence microscopy NOE: and Avidin iotin Complex (AC) can be integrated into systems currently using the AC method, either before or after the AC step. 1 Ab 10

11 Detection In Situ Hybridization Chromogenic with - Amplification and Visualization Procedures After signal amplification, the deposited biotin can be directly visualized by any of the following methods: Addition of - followed by horseradish peroxidase catalyzed chromogen Chromogenic with - Addition of - followed by alkaline phosphatase catalyzed chromogen luorescence with -luorophore Addition of -luorophore (-luorescein) (-Coumarin) (-exas Red ) viewed by fluorescence microscopy Probe Labeling Option Ab Ab D 11

12 here s a system PerkinElmer offers a variety of systems to enable every lab to benefit from the sensitivity enhancements of this outstanding technology. You ll maximize the difference that makes in your IHC and ISH applications when you select a system based on your research requirements and the experience of your lab. irst, 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. hen, based on your experience, choose one of three formats: Complete Systems: complete tyramide signal amplification kits include labeled tyramide, amplification diluent, streptavidin, blocking reagent and comprehensive instruction manuals. Additional antibodies, conjugates, and chromogens may be required for your optimal protocol and may be purchased separately. Complete Plus Systems: tyramide signal amplification kits that provide 10 to 20 times the sensitivity enhancement of regular systems. Reagent Packs: stand-alone amplification reagents that provide researchers experienced with the flexibility to develop their own protocols. Mouse rain, 20x magnification, 15 second exposure Conventional detection with fluorescein conjugated 2 antibody. Dilution 1:100 Standard fluorescein. Dilution 1:10,000 Plus fluorescein. Dilution 1:1,000,000 12

13 for your laboratory Complete Systems luorescence Systems Used for direct fluorescence detection in IHC and ISH applications. While not as sensitive as Plus systems, allow significant reductions in antibody or probe with necessary sensitivity in many applications. A iotin 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. IHC detection of CD3 antigen in serial sections of paraffinembedded human tonsil. (A) Rabbit anti-cd3 1:400, biotinylated anti-rabbit, AC, with DA chromogenic detection. () iotin: Rabbit anti-cd3 1:400, biotinylated anti-rabbit, -, biotinyl tyramide, AC, with DA chromogenic detection. (Courtesy of. van den erg and A. de Koning, Dept. Pathology, AMC, Amsterdam, he Netherlands.) Complete Plus Systems Plus luorescence 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, MR, coumarin, Cyanine-3, or Cyanine-5. Embryonic mouse brain. Histone H4 mrna ISH, using Plus Cyanine 3 and M2 immunoreactivity (IHC) with Plus luorescein System. Plus luorescence 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 fluorescence detection or exploring multi-target detection is easy with our Plus luorescence Palette System, a trial-sized kit with four different fluors packaged together. Each fluor has sufficient reagent for slides. Multi-target detection in the same sample. Detection of Arg- Vasopressin and Oxytocin in rat brain using Cyanine 3 and Cyanine 5, respectively. 13

14 with the flexibility to match your research and application needs. 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. he 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. Reagent Packs Reagent Packs are stand-alone tyramide () kits for signal amplification that provide flexibility to researchers for protocol development. Researchers must supply their own blocking buffer, and other reagents as needed. If you have not used Signal Amplification reagents before, we strongly suggest purchasing one of our complete systems, which include a set of quality controlled reagents optimized to work well together. Use versatile, flexible to enhance sensitivity in many applications makes a difference in the sensitivity of any application using an antibody or gene probe that allows the addition of into its protocol: ELI: amplify signal in microplate-based assays, greatly increasing sensitivity or reducing the use of antibodies. Use our convenient ELAS ELI Amplification System. Protein microarrays: detect low abundance proteins in differential protein expression experiments. Use our AUOMAED Kits with our ProteinArray Workstation, a highly flexible, fully automated system for processing multiple protein microarrays. Genomic microarrays: apply to your cdna differential gene expression studies using our MICROMAX Labeling and Detection Kits. is compatible with all common detection methods: Enzymatic AC (avidin-biotinylated complex) P (peroxidase anti-peroxidase) (alkaline phosphatase anti-alkaline phosphatase) Chromogenic CIP/N (using alkaline phosphataseconjugated secondary antibody or avidin) DA (using second application of conjugated secondary antibody or avidin) Electron Microscopy (EM) Gold conjugate Immunogold detection systems luorescence (Direct and Indirect) 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 to your protocols. Contact us at (800) or (+1) , or by at techsupport@perkinelmer.com or techsupport.europe@perkinelmer.com. 14

15 Ordering Information luorescence Systems Product No. of Slides Product No. luorescein System NEL701A001K NEL701001K MR System NEL K Coumarin System NEL703001K Cyanine 3 System NEL704A001K Cyanine 5 System* NEL705A001K iotin Systems Product No. of Slides Product No. iotin System NEL700A001K NEL700001K Plus luorescence Systems Product No. of Slides Product No. Plus luorescein System NEL741001K NEL741001K Plus MR System NEL742001K NEL742001K Plus Cyanine 3 System NEL744001K NEL744001K Plus Cyanine 5 System* NEL745001K NEL745001K Plus luorescence Palette Systems Product No. of Slides Product No. Plus Cyanine 3/Cyanine 5 System* NEL752001K Plus Cyanine 3/luorescein System NEL753001K Plus Cyanine 5/luorescein System* NEL754001K Plus luorescein/mr System NEL756001K Plus luorescence Palette System (contains one each of luorescein, MR, Cyanine 3 and Cyanine 5)* NEL760001K Plus DNP Systems (iotin-free) Product No. of Slides Product No. Plus DNP () System NEL746001K NEL746A001K Plus DNP () System NEL747001K NEL747A001K Reagent Packs Product No. of Slides Product No. iotin yramide Reagent Pack EA 1,000 3, EA luorescein yramide Reagent Pack EA 500 1, EA MR yramide Reagent Pack EA Cyanine 3 yramide Reagent Pack A001EA EA Cyanine 5 yramide Reagent Pack* A001EA *Cyanine 5 tyramides cannot be visualized with most digital cameras. 15

16 Complementary Products for Use with hese high quality conjugates, chromogens and secondary antibodies can be used with Assay Systems and Reagent Packs. Chromogens & Conjugates Product CIP/N ( substrate) DA ( substrate) Antifluorescein- Antifluorescein- Streptavidin luorescein Streptavidin exas Red Streptavidin Coumarin Streptavidin- Streptavidin- Product No. NEL937001PK NEL938001EA NE709001PK NE710001EA NEL720001EA NEL721001EA NEL722001EA NEL750001EA NEL751001EA Secondary Antibodies Antibody Label Size Concentration Product No. Anti-human IgG (goat)* 1 ml 1 mg/ml NE801001EA Anti-human IgG (goat)* 1 ml 1 mg/ml NE802001EA Anti-human IgG (goat) iotin 0.5 mg Lyophilized NE803001EA Anti-mouse IgG (goat) 1 ml 1 mg/ml NE821001EA Anti-mouse IgG (goat) 1 ml 1 mg/ml NE822001EA Anti-mouse IgG (goat) iotin 0.5 mg Lyophilized NE823001EA Anti-rabbit IgG (goat) 1 ml 1 mg/ml NE811001EA Anti-rabbit IgG (goat) 1 ml 1 mg/ml NE812001EA Anti-rabbit IgG (goat) iotin 0.5 mg Lyophilized NE813001EA *ovine serum albumin added as a stabilizer. technology from PerkinElmer the difference is clear! Add 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. or more information, visit phone (800) or (+1) , or contact your local sales representative. or a complete listing of our global offices, visit PerkinElmer Life and Analytical Sciences 710 ridgeport Avenue Shelton, C U Phone: (800) or (+1) or a complete listing of our global offices, visit PerkinElmer, Inc. All rights reserved. he PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc., ProteinArray, MICROMAX and ELAS are trademarks or registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. exas Red is a registered trademark of eletype 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 _01 Printed in U