See ihC taRgetS moRe CLeaRLy TSA TSA Signal Amplification for immunohistochemistry (ihc) Immunocytochemistry (ICC), Immunofluoresence (IF) TSA for IHC See what you’ve been missing The extraordinary sensitivity of the Tyramide Signal Amplification (TSA™) kits from PerkinElmer lets you see previously undetectable levels of protein and nucleic acid. The resolution is remarkable. And with multi-target detection, you can get more information from each experiment. It’s clear to see. TSA makes it easy to gain valuable insight from your immunohistochemistry (IHC) and immunocytochemistry (ICC) results. • See previously undetectable low-abundance targets • Achieve outstanding resolution and clarity • Conserve precious antibody while improving specificity • Add to current protocol with minimal disruption • Eliminate background problems with TSA’s biotin-free formats • Investigate co-localization with multi-target detection kits TSA is ideal for use with PerkinElmer's Cellular Imaging and Analysis solutions. Reach never-before-seen levels of detection TSA can increase sensitivity up to 1000-fold. Detection of CD31 (PECAM-1) Mouse Embryo Heart Tissue. Sensitivity comes with excellent resolution – a combination critical to localizing lowabundance and previously unsuspected targets. It adds up to never-before-seen levels of detection. Standard IHC with Anti-CD31 dilution 1:100, secondary antibody fluorophore labeled and counterstained with DAPI. TSA IHC with Anti-CD31 dilution 1:100, fluorescent detection (fluorescein) and counterstained with DAPI. Courtesy of Bin Zhao at Harvard Stem Cell Institute Use TSA Plus to enhance sensitivity 10-20-fold over standard TSA. www.perkinelmer.com/tsa Improve Sensitivity and Resolution Dramatically TSA improves resolution while enhancing sensitivity. Covalent labeling means sharper images with clearer results. Comparison of direct immunofluorescent staining and TSA-enhanced direct fluorescent staining of CMVinfested cells. Direct fluorescent staining. Direct fluorescent staining enhanced by TSA. IHC detection of CD3 antigen in serial sections of paraffin-embedded human tonsil. Rabbit anti-CD3 1:400, biotinylated anti-rabbit, ABC, with DAB chromogenic detection. Courtesy of F. van den Berg and A. de Koning, Dept. Pathology, AMC, Amsterdam, the Netherlands TSA biotin: rabbit anti-CD3 1:400, biotinylated anti-rabbit, SA-HRP, biotinyl tyramide, ABC, with DAB chromogenic detection. Reduce Primary Antibody Consumption While Improving Specificity Use up to 1000-fold less primary antibody while reducing non-specific detection and the cost of your assay. Comparison of standard IHC and TSA in the detection of Epstein-Barr Virus – Hodgkins Lymphoma Tissue. Standard IHC with Anti-EBV dilution 1:25 and fluorescent detection with conjugated antibody. TSA IHC with Anti-EBV dilution 1:25,000 and fluorescent detection. Courtesy of R. Von Wasielewski and S. Gignac, Pathologisches Institut de Medizinischen Hochscule, Hannover, Germany Mouse Brain, 20x magnification, 2-second exposure Conventional detection with Cyanine 3 conjugated 2º antibody. Dilution 1:1,000. Standard TSA Cyanine 3. Dilution 1:100,000. TSA Plus Cyanine 3. Dilution 1:10,000,000. 4 hoW does tsa WoRK foR ihc/if/icc? 3. incubate with tsa reagent (3-10 min). hRp catalyzes formation of tsa free radicals. T Ag TT TT T T Ag T Ag T T Ag HRP T T T T T T TT T T T T T T T T T T T HRP T T T T T HRP T T T H2O T T T H2O2 4. tsa free radicals form covalent bonds with tyrosine residues proximal to hRp. unbound tsa radicals form dimers that are washed away. T 2. introduce hRp T 1. incubate with primary antibody adding tsa to ihc / icc / immunofluoRescence assays Antigen Primary antibody Introduction of HRP TSA possibilities Detection options direct fluorescence F T HRP SA B • Coumarin (ex. 402 nm, em. 443 nm) • Fluorescein (ex. 494 nm, em. 517 nm) • TMR (ex. 550 nm, em. 570 nm) Hapten-labeled secondary antibody • Cyanine 5 (ex. 648 nm, em. 667 nm) T DNP T B T FITC HRP HRP-conjugated secondary antibody (other options include HRP polymer conjugates, ABC) • Cyanine 3 (ex. 550 nm, em. 570 nm) SA, Anti-FITC HRP conjugate… perkinelmer, inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com for a complete listing of our global offices, visit www.perkinelmer.com/contactus Copyright ©2009-2010, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 008635A_01 Printed in USA Sep. 2010 chromogenic • Streptavidin-HRP or AP plus chromogen of choice • Anti-DNP-HRP or AP with chromogen of choice • Anti-fluorescein-HRP or AP with chromogen of choice indirect fluorescence • Streptavidin-fluorophore conjugate • Anti-DNP-fluorophore conjugate • Anti-fluorescein-fluorophore conjugate