6.2 Tyramide Signal Amplification (TSA) Technology

Transcription

1 6.2 Tyramide Signal Amplification (TSA) Technology Principles of Tyramide Signal Amplification Figure 6.6 Schematic representation of TSA detection methods applied to immunolabeling of an antigen. Detection of the antigen by a primary antibody and a horseradish peroxidase labeled secondary antibody, resulting in localized deposition of dyeor hapten-labeled tyramide (Stage 1). Further dye deposition, and therefore higher levels of signal amplification, can be generated by detection of dye deposited in stage 1 by a horseradish peroxidase labeled anti-dye primary antibody (Stage 2). Tyramide signal amplification (TSA) 1 sometimes called CARD, for Catalyzed Reporter Deposition is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. 2 6 The TSA method has been reported to increase the sensitivity by up to 100-fold compared to conventional avidin biotinylated enzyme complex (ABC) procedures. 4,7 13 Molecular Probes is committed to the extensive development of TSA in combination with our proprietary dyes and other detection technology in order to achieve the ultimate in high-resolution signal amplification technology in cellular and tissue applications. 1 We expect to develop kits based on TSA technology for multicolor labeling of targets in fluorescence in situ hybridization (FISH), immunocytochemistry, immunohistochemistry, cell tracing, enzyme-amplified flow cytometry and detection of very low abundance receptors. For multiparameter detection of targets in either live or fixed cells or tissues, TSA can be combined with several of our other important technologies, including ChromaTide nucleotides, ULYSIS and ARES Nucleic Acid Labeling Kits (Section 8.2), primary and secondary antibodies, avidin and lectin conjugates (Chapter 7), Enzyme- Labeled Fluorescence (ELF, Section 6.3), cytoskeletal stains (Chapter 11), organelle probes (Chapter 12), cell tracers and proliferation markers (Chapter 14, Chapter 15) and receptor probes (Chapter 16). Our proprietary Zenon One Mouse IgG 1 Labeling Kits (Section 7.2, Table 7.1) permit the facile preparation of dye- or enzyme-labeled mouse IgG 1 in quantitative yields on even sub-micrograms of the primary antibody (Figure 7.32). In addition, the Zenon One Kits enable multiple mouse monoclonal antibodies to be combined for use in the same experiment. Our Zenon One Horseradish Peroxidase Mouse IgG 1 Labeling Kit (Z-25054) is of particular utility when used in combination with TSA technology. TSA labeling is a combination of three (or four) elementary processes (Figure 6.6) that typically comprise: Binding of a probe to the target via immunoaffinity (proteins) or hybridization (nucleic acids) followed by secondary detection of the probe with an HRP-labeled antibody or streptavidin conjugate. Peroxidase conjugates of other targeting proteins such as lectins and receptor ligands are likely to be suitable for labeling targets, as is endogenous peroxidase activity. 14,15 Unconjugated HRP is also useful as a neuronal tracer; its use in combination with TSA is demonstrated in Figure Activation of multiple copies of a labeled tyramide derivative by the HRP. Most often a fluorescent or biotinylated tyramide derivative has been used; however, labeling with Figure 6.7 Coupling of Alexa Fluor 488 tyramide to protein tyrosine side chains via peroxidase-mediated formation of a tyrosine adduct. 152 Chapter 6 Ultrasensitive Detection Technology

2 other hapten-conjugated tyramides 16,17 or with polymeric reagents, including tyramide-conjugated gold particles has also been reported. 18 Covalent coupling of the resulting highly reactive, short-lived tyramide radicals to residues (principally the phenol moiety of protein tyrosine residues) in the vicinity of the HRP target-interaction site, resulting in minimal diffusion-related loss of signal localization (Figure 6.7). A unique application uses trapping of a dye-labeled tyramide by phenoxyl radicals that are generated from tyrosine residues of proteins as the result of formation of reactive oxygen species (ROS, Section 19.2) in live cells. 19 In direct TSA, the fluorescent signal can be immediately detected, resulting in both excellent spatial resolution (Figure 6.8, Figure 6.9) and high signal intensity. When using a hapten-labeled tyramide such as biotin-xx tyramide, the easily reversible DSB-X biotin tyramide or DNP-X tyramide (see below), a subsequent detection step is required using a bioconjugate that recognizes the hapten. This second detection step can include a dye-labeled hapten recognizer such as a fluorescent streptavidin (Section 7.6) or a fluorescent anti-hapten antibody (Section 7.4). Alternatively, the hapten-labeled tyramide can be detected using an alkaline phosphate or HRP-labeled hapten recognizer in conjunction with a fluorogenic or chromogenic substrate (Figure 6.10) resulting in another enzyme-amplified detection step. Chemiluminescent detection of an HRPdeposited biotin tyramide has also been reported. 20 The streptavidin conjugate of NANOGOLD 1.4 nm gold clusters (N-24918, Section 7.6) has been used to make biotin tyramide conjugates visible in light and electron microscopy. 21 Presumably the Alexa Fluor conjugates of FluoroNanogold (Section 7.2, Section 7.6) can be used with haptenlabeled tyramides for correlated fluorescence, light and electron microscopy studies. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label translates into practical benefits, namely ultrasensitive detection of low- Figure 6.8 Golgi in HeLa cells detected with Alexa Fluor 546 tyramide. Cells were fixed and permeabilized, then labeled with anti human golgin-97 (A-21270) and detected using HRP-conjugated goat anti mouse IgG antibody and Alexa Fluor 546 tyramide, which are components of our TSA Kit #3 (T-20913). The nuclei were counterstained using DAPI (D-1306, D-3571, D-21490). The images were acquired using filters appropriate for DAPI and Alexa Fluor 546. Figure 6.9 Tyramide signal amplification of immunofluorescent staining in mouse brain sections. Mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate buffer. 30 µm serial sections were cut in a freezing microtome and transferred to phosphate-buffered saline. Free-floating sections were incubated with 1% hydrogen peroxide to quench endogenous peroxidase activity, blocked in 5% normal goat serum, then stained with a rabbit polyclonal antibody to calbindin D-28K (Chemicon) at a 1:1000 dilution. After washing, sections were incubated with Alexa Fluor 488 goat anti rabbit IgG antibody (A-11008) at 5 µg/ml (left panel) or HRP goat anti rabbit IgG antibody at 1 µg/ml, followed by Alexa Fluor 488 tyramide (in TSA Kit #12, T-20922; right panel). Sections were washed, mounted on slides, coverslipped with ProLong antifade reagent (in Kit P-7481) and imaged under identical conditions (10 magnification, 250 millisecond exposure) using a bandpass filter set appropriate for fluorescein (FITC). Figure 6.10 Bovine pulmonary arterial endothelial (BPAE) cells were labeled with 5-bromo-2 -deoxyuridine (BrdU, B-23151) applied at a concentration of 10 µm for 30 minutes. After fixation with 4% formaldehyde in phosphate-buffered saline for 30 minutes, chromatin was denatured by treatment with 2 M HCl for 20 minutes. Incorporated BrdU was detected with mouse monoclonal anti-bromodeoxyuridine antibody (A-21300) followed by HRPconjugated goat anti mouse IgG antibody and Oregon Green 488 tyramide (TSA Kit #9, T-20919). Tyramide labeling was further amplified and converted for visualization by bright-field microscopy by detection of the Oregon Green 488 dye hapten using the HRP conjugate of anti-fluorescein/oregon Green antibody (A-21253) and diaminobenzidine (DAB) staining. Both nuclear and non-nuclear (presumably mitochondrial) incorporation of BrdU is clearly visible in the resulting image. Section

3 abundance targets in fluorescence in situ hybridization, 4,22,23 immunohistochemistry, 7,24 neuroanatomical tracing, 8,25 and other applications. For instance, we have utilized TSA and Alexa Fluor 488 tyramide to detect expression of low-abundance epidermal growth factor (EGF) and estrogen receptors by flow cytometry with far greater sensitivity than can be obtained using a directly labeled EGF probe (Figure 6.11) or fluorophore- or hapten-labeled antibodies to the estrogen receptor (Figure 6.12). Application of TSA resulted in significantly increased detectability of estrogen receptors in urinary bladder carcinomas compared to conventional immunohistochemical analysis. 26 We have already developed 36 TSA kits that combine the versatile tyramide signal amplification technology with seven of our high-performance Alexa Fluor dyes, our Oregon Green 488 dye, our Pacific Blue dye, or with biotin-xx tyramide, DSB-X biotin tyramide or 2,4-dinitrophenyl (DNP)-X tyramide (Table 6.1). We anticipate developing several different applications for TSA technology directed at ultrasensitive detection of selected targets in cells and tissues. Immunohistochemical Detection using TSA Figure 6.11 Detection of epidermal growth factor (EGF) receptors directly or with signal amplification. Cells expressing high (A431 cells, Panel A) and low (NIH 3T3 cells, Panel B) levels of EGF receptors were either directly labeled with the preformed Alexa Fluor 488 complex of biotinylated epidermal growth factor (E-13345, blue) or indirectly labeled with biotinylated EGF (E-3477) followed by either Alexa Fluor 488 streptavidin (S-11223, green) or HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (purple), components of our TSA Kit #22 (T-20932). In immunohistochemical applications (Figure 6.9, Figure 6.13), sensitivity enhancements derived from TSA allow primary antibody dilutions to be increased up to a 1:1,000,000 antibody dilution was possible in one reported case, 11 although a 5- to 50- fold increase over the normal dilution factor is more common 10 in order to reduce nonspecific background signals. 8 Additionally, the strong signal amplification provided by the TSA method can overcome relatively high autofluorescence of cells and tissues. 7 Thus, TSA detection can be applied to a variety of immunohistochemical specimen preparations, including crytostat sections, formaldehyde-fixed paraffin-embedded sections, plastic-embedded sections and cultured cells. In a very unique application, the significantly lower detection threshold of TSA compared to fluorescent secondary antibodies allows detection of two targets by primary antibodies raised in the same host species, without substantial crosstalk between the signals. The first target is detected using TSA and a primary antibody concentration that is so low that it is essentially undetectable by fluorescent secondary antibodies. The second target is then detected by conventional secondary immunofluorescence labeling. 24,27 Our Zenon One technology (Section 7.2) provides an alternative method for using multiple mouse IgG 1 antibodies in a single experiment. Furthermore, because TSA and diaminobenzidine (DAB) oxidation are both peroxidase-mediated reactions, TSA is readily adaptable for correlated fluorescence and electron microscopy studies. 28,29 Table 6.1 Tyramide signal amplification (TSA) kits. 154 Chapter 6 Ultrasensitive Detection Technology

4 Fluorescence In Situ Hybridization using TSA The increased sensitivity afforded by TSA (Figure 6.14, Figure 6.15, Figure 8.83) can be critically important for detection of relatively short oligonucleotide probes and low-abundance mrnas by fluorescence in situ hybridization (FISH). 4,30 Cosmid detection in formalin-fixed, paraffin-embedded sections is cumbersome, and the ability to use smaller cosmid probes of less than 1000 bases and TSA detection technology is likely to be an important technique for FISH. 22 TSA is also faster than traditional FISH detection schemes, allowing definitive results to be obtained within a single day. In addition, a two-stage amplification method for ultrasensitive mrna detection has been reported that combines TSA detection of biotinylated riboprobes with alkaline phosphatase mediated fluorescence generation using Molecular Probes unique ELF 97 phosphatase substrate 31 (Section 6.3). However, TSA is not a panacea for FISH sensitivity problems. Because both specifically and nonspecifically bound probe signals are amplified, TSA will not compensate for suboptimal hybridization conditions. Optimal probe concentrations are typically 2 10 fold lower for TSA-detected FISH than for conventional immunocytochemical detection procedures, again saving on the cost of expensive hybridization probes. 23 Typically, FISH probes are labeled by indirect methods that use streptavidin- or antibody-conjugated HRP. Techniques for direct labeling of oligonucleotide probes have been developed to eliminate background signals due to nonspecific binding of peroxidase conjugates. 5,32,33 As with other detection systems, several probes can be hybridized simultaneously to identify multiple targets. Signal development using multicolored fluorescent tyramides must be carried out sequentially, with a peroxidase inactivation step between each TSA reaction to prevent crosstalk 23 (Figure 6.14). TSA amplification followed by peroxidase inactivation through mild acid treatment with 0.01 M HCl for 10 minutes at room temperature 34 and then reapplication of TSA using a fluorescent tyramide of a different fluorescent color has been used for at least triple-labeled in situ hybridization. 5,34 Molecular Probes TSA Detection Kits Our TSA detection kits (Table 6.1) offer 36 different combinations of Alexa Fluor dye, Oregon Green 488 dye, Pacific Blue dye, biotin-xx, DSB-X biotin or 2,4-dinitrophenyl (DNP-X) labeled tyramides and streptavidin, anti mouse IgG antibody or anti rabbit IgG antibody conjugates of HRP. Each kit provides sufficient materials to stain slide preparations and includes the following components: Alexa Fluor dye, Oregon Green 488 dye, Pacific Blue dye, biotin-xx, DSB-X biotin or DNP-X labeled tyramide An HRP-conjugated secondary antibody or streptavidin Amplification reaction buffer H 2 O 2 reaction additive A blocking reagent A staining protocol Our fluorescent dye and hapten-labeled tyramides are not available as standalone reagents. Our Zenon One Horseradish Peroxidase Mouse IgG 1 Labeling Kit (Z-25054, Section 7.2) provides an alternative for rapid labeling of mouse (or rat) monoclonal antibodies of the IgG 1 isotype that have intact Fc portions. Some crossreactivity of the Zenon One reagents with other isotypes of mouse antibodies may be observed. The Zenon One labeling reagent in this kit makes it possible to quantitatively label even submicrograms of a primary antibody with HRP immediately before it is applied to the sample. The Zenon One reagent in this kit can replace the HRP goat anti mouse IgG antibody conjugates in any of our TSA kits that Figure 6.12 Enhancement of estrogen receptor detection sensitivity by tyramide signal amplification (TSA). SKBR3 cells with characteristically low levels of estrogen receptor expression were fixed, permeabilized and treated with H 2 O 2 to inhibit endogenous peroxidase activity. A mouse anti human estrogen receptor monoclonal antibody (Chemicon) was labeled with the Alexa Fluor 488 dye or with biotin using our Zenon One Alexa Fluor 488 Mouse IgG 1 Labeling Kit (Z-25002) and Zenon One Biotin-XX Mouse IgG 1 Labeling Kit (Z-25052), respectively. Detection of estrogen receptors using the labeled antibodies was performed on a Becton Dickinson FACScan flow cytometer with excitation at 488 nm. The cellular fluorescence intensity histograms represent detection with Alexa Fluor 488 dye labeled antibodies (blue), biotinylated antibodies coupled to Alexa Fluor 488 streptavidin (S-11223, green) and biotinylated antibodies coupled to HRP streptavidin and Alexa Fluor 488 tyramide (TSA Kit #22, T-20932; orange). The red histogram represents unstained cells. Figure 6.13 Immunohistochemical detection using tyramide signal amplification. A transverse section of fixed zebrafish retina was probed with mouse monoclonal FRet 34 and subsequently developed for visualization using HRPconjugated goat anti mouse IgG antibody and Alexa Fluor 488 tyramide, which are supplied in our TSA Kit #2 (T-20912). The section was counterstained with the blue-fluorescent Alexa Fluor 350 wheat germ agglutinin (W-11263) and the far red-fluorescent TOTO-3 nuclear stain (T-3604). Section

5 contain that conjugate. The Zenon One Horseradish Peroxidase Mouse IgG 1 Labeling Kit contains sufficient reagents to prepare HRP conjugates of 50 µg of any whole mouse IgG 1. Applying TSA Technology to Cells and Tissues Our TSA Bibliography The TSA technology has been known for several years. 2,35 Many papers have been published that have utilized biotin tyramide for indirect labeling of targets or various fluorescent tyramide conjugates for direct labeling of targets. Direct labeling methods have the considerable advantage of saving a second step in the detection scheme. Labeling of targets by fluorescent tyramides has the further advantage over biotin tyramide of avoiding amplification of endogenous biotin in cells and tissues, such as we have observed in mitochondrial staining with streptavidin conjugates in the absence of a biotinylated probe (Figure 12.29). We have compiled an extensive bibliography that lists several suggested applications; however, the biotin tyramide used in all previous references did not have the additional 14-atom spacer that we utilize in our biotin-xx tyramide to make the probe more accessible to avidin conjugates. Furthermore, none of the specific fluorescent dyes or haptens in these earlier papers are available in kits provided by Molecular Probes, so the specific methods described in these references should be considered guides rather than definitive protocols and results using our TSA reagents may differ from those reported. However, in our experience, our Alexa Fluor 488 tyramide derivative (Table 6.1) provides greater signal and significantly greater photostability than fluorescein tyramide, and the other Alexa Fluor tyramides, Oregon Green 488 tyramide and Pacific Blue tyramide also yield intense staining of targets. Detection of Biotin-XX Tyramide, DSB-X Biotin Tyramide and Hapten-Labeled Tyramides When utilizing a hapten-labeled tyramide, such as biotin-xx tyramide or the readily reversible hapten, DSB-X biotin (Section 7.6), for TSA in an indirect labeling technique, it is necessary to provide a signal-generation reagent or scheme. A fluorescent tyramide such as our Oregon Green tyramide can also be utilized as a hapten for an antibody to the fluorophore in a subsequent amplification scheme. Various reagents and reagent combinations have been reported for detecting enzyme-deposited biotin tyramide or fluorescein tyramide that should be equally suitable for use with our biotin-xx tyramide, DSB-X biotin tyramide, DNP- X tyramide or Oregon Green 488 tyramide, including: The streptavidin conjugate of alkaline phosphatase (S-921, Section 7.6) or the rabbit anti-fluorescein/oregon Green antibody conjugate of alkaline phosphatase 36 (A-21251, A-21252; Section 7.4), in combination with NBT/BCIP 37,38 (N-6495, B-6492; Section 10.3) or ELF 97 phosphate 31 (E-6588, Section 6.3). Our antibodies to the DNP chromophore, which are described in Section 7.4 (Table 7.14). The streptavidin conjugate of HRP (S-911, Section 7.6) or the rabbit anti-fluorescein/oregon Green antibody conjugate of HRP 27,30,39 41 (A-21253, A-21254; Section 7.4) in combination with traditional chromogenic peroxidase substrates such as diaminobenzidine (DAB) 42,43 (Figure 6.10). Fluorescent conjugates of avidin or streptavidin, several of which are listed in Table 7.17 and Section 7.6. Our Alexa Figure 6.14 In situ hybridization of α-satellite probes to human chromosomes 1, 15 and 17 detected by tyramide signal amplification. α-satellite probes to chromosomes 1, 15 and 17 were labeled by nick translation with ChromaTide biotin- 11-dUTP (C-11411), ChromaTide Texas Red-12-dUTP (C-7631) and ChromaTide Oregon Green dUTP (C-7630), respectively. Following simultaneous hybridization of all three probes, the biotinylated chromosome 1 probe was detected with HRP streptavidin and Alexa Fluor 546 tyramide (TSA Kit #23, T-20933). HRP activity from this first TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. The Oregon Green 488 dye labeled chromosome 17 probe was then detected with anti-fluorescein/oregon Green antibody (A-6421) followed by HRP-conjugated goat anti mouse IgG antibody and Alexa Fluor 594 tyramide (TSA Kit #5, T-20915). HRP activity from this second TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. The Texas Red dye labeled chromosome 15 probe was then detected with rabbit anti Texas Red antibody (A-6399) followed by HRPconjugated goat anti rabbit IgG antibody and Alexa Fluor 488 tyramide (TSA Kit #12, T-20922). After counterstaining with Hoechst (H-1398, H-3569, H-21491), images were acquired using filters appropriate for DAPI, FITC, TRITC and the Texas Red dye. Figure 6.15 Digital image analysis comparison of in situ hybridized biotinylated α-satellite probes detected using TSA Kit #23 (A-21003) with HRP streptavidin and Alexa Fluor 546 tyramide (right) or Alexa Fluor 546 streptavidin (S-11225, left). Both images were converted to pixel intensity values using MetaMorph software (Universal Imaging Corporation) and transferred to a Microsoft Excel spreadsheet for plotting. Alexa Fluor 546 dye and DAPI (counterstain) intensity values are shown in red and blue, respectively. Alexa Fluor 546 dye intensity values below 35% of maximum were omitted for clarity. 156 Chapter 6 Ultrasensitive Detection Technology

6 Fluor 488, Alexa Fluor 594 and Texas Red conjugates of antifluorescein/oregon Green antibody (Section 7.4) can potentially be used to further amplify the signal (Figure 6.6) or shift the emission of the green-fluorescent Oregon Green 488 tyramide to red fluorescence. Diaminobenzidine (DAB) Histochemistry Kits (D-22185, D-22186, D-22187; Section 7.3) for direct use with biotin-xx tyramide and DSB-X biotin tyramide or conversion of fluorescent signals to permanent staining. NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies (Section 7.3) and streptavidin (Section 7.6) for target localization using a combination of light and electron microscopy. 44 The streptavidin conjugate of Captivate ferrofluid (C-21476, Section 7.6), which can be utilized to efficiently separate lowabundance cellular targets amplified by biotin-xx tyramide or DSB-X biotin tyramide and the TSA technology. Binding of DSB-X tyramide labeled targets to the Captivate ferrofluid is reversible under extremely mild conditions, potentially permitting isolation of fully viable rare cells using the unique Captivate microscope-mounted magnetic yoke assembly (C-24700, Section 24.3) and disposable sample chambers (C-24701, Section 24.3), which have been specially designed for use with the Captivate ferrofluid conjugates (Figure 7.62). Some selected applications that have been reported for biotin tyramide that should work at least as well with our biotin-xx tyramide include: Fluorescence in situ hybridization in paraffin wax embedded sections of colon tumor cells 45 mrna detection of low-abundance cytokine genes using direct HRP conjugates of hybridization probes 5,32 Detection of low copy number sequences of human papillomavirus 46,47 Combination of mrna in situ hybridization and immunohistochemical detection 48 Use of relatively short probes for DNA and mrna FISH 49,50 In situ PCR amplification of HIV-1 DNA in brain tissue, 37 HIV-1 p24 antigen detection in paraffin sections 51 and detection of HIV-1 viral DNA integration sites using FISH 52 Detection of low-abundance targets with mrna probes in cryosections 30 Generation of a color bar-code for genes with the Fiber-FISH technique 22,53 In situ hybridization in semithin plastic sections 39 Demonstration of macrophage in histochemical sections 54 Immunofluorescent labeling of GABA A receptor subunits in the human brain 55 β-galactosidase (LacZ) detection in paraffin-embedded tissue sections 56 Localization of the glial cell line derived neurotrophic factor (GDNF) and its functional receptor in the dorsal root ganglion of rat nerves 57 Endosome labeling using HRP-conjugated transferrin and biotin tyramide 58 Detection of the distribution of nitric oxide synthase in developing embryonic rat brain, 43 blood vessels of rat brain 59 and bovine aorta 60 Amplified detection of low-abundance targets in cultured neurites 58 Anterograde tracing using HRP-conjugated lectins 25,61 Conversion of TSA to an electron microscopy detection method 29 Previously cited applications of various fluorescent tyramides that are not available from Molecular Probes include: Fluorescence in situ hybridization 5,34 Double- and triple-color immunofluorescence studies using unconjugated primary antisera raised in the same species 24,62 Detection of chromosome-specific repeat sequences 63 Escherichia coli detection and speciation in water with a biotinylated peptide nucleic acid 64 Detection of cyanobacteria in a highly autofluorescent sample with a singly labeled oligonucleotide probe 65 Flow cytometric characterization of labeled bacteria 66 Localization of the HuC and HuR chromosomes by FISH 67 Simultaneous detection of two neuropeptide receptors 41,68 Immunocytochemical detection of low-level incorporation of 5-bromo-2 -deoxyuridine (BrdU, B-23151; Section 15.4) in dividing cells 7 (Figure 6.10) Localization of a scarce leptin receptor using TSA in combination with ELF 97 phosphate 31 (Section 6.3) Anti-fluorescein/Oregon Green antibody conjugates of HRP or alkaline phosphatase (Section 7.4, Table 7.13) have been used with a fluorescein tyramide to detect: Embryonic gene expression at the cellular level by FISH 69 An mrna probe for a calcium transporter protein 36 A somatostatin receptor protein 40 Tissue antigens with a 10- to 100-fold increase in sensitivity over conventional staining methods 27 mrna in paraffin sections of organotypic multicellular spheroids 70 Double and Sequential Amplification with TSA An exciting possibility for TSA that we are currently exploring is the double- or even triple-application of our signal amplification technologies by using sequential rounds of TSA 71 (Figure 6.6) or TSA in combination with our ELF technology 31 (Section 6.3) or with the permanent histochemical stains DAB (for HRP) or NBT/BCIP (for alkaline phosphatase). For instance, in the first round biotin-xx tyramide can be deposited on a target using one of our biotin-xx tyramide TSA Kits (Table 6.1). In a subsequent step, the peroxidase conjugate of streptavidin that is used in TSA Kit #21 (T-20931) is used again but this time in combination with an Alexa Fluor tyramide, Oregon Green 488 tyramide, Pacific Blue tyramide or another round of biotin-xx tyramide. Presumably this amplification can be continued for at least a third round, although some loss of spatial resolution may result. Biotin tyramide that has first been deposited at the binding site of a biotin-labeled riboprobe using the streptavidin conjugate of HRP has been further amplified with the streptavidin conjugate of alkaline phosphatase (S-921, Section 7.6) in conjunction with ELF 97 phosphate for the ultrasensitive detection of a scarce leptin receptor mrna. 31 In Section

7 another example 72 demonstrating the versatility of the TSA technology, several labeling technologies were combined to detect the HIV-1 virus: Hybridization with a 15-base peptide nucleic acid probe labeled with a single fluorescein dye Complexation with an HRP conjugate of anti-fluorescein antibody Incubation with biotin tyramide Incubation with the streptavidin conjugate of HRP Re-incubation with biotin tyramide Detection with the Alexa Fluor 488 conjugate of streptavidin (S-11223, Section 7.6) Alternatively, for detection by light microscopy the sample was incubated with the streptavidin conjugate of HRP in conjunction with DAB instead of with Alexa Fluor 488 streptavidin. DAB Histochemistry Kits The use of HRP for enzyme-amplified immunodetection commonly referred to as immunoperoxidase labeling is a wellestablished standard histochemical technique. 73,74 The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse (D-22185) and rabbit (D-22186) IgG primary antibodies and biotinylated antibodies and tracers (D-22187). Each kit contains: An HRP-labeled secondary antibody or streptavidin conjugate The DAB substrate H 2 O 2 reaction additive Reaction buffer Blocking reagent A detailed staining protocol Each kit provides sufficient materials to stain approximately 200 slides. Additional Tips on Using TSA Technology Use of the TSA technology is not without its precautions. Among these is the possibility of endogenous peroxidase activity in certain cells, especially eosinophils. 15 This activity can be at least partially blocked by incubation with 0.3 3% hydrogen peroxide for about 60 minutes. Second, when using biotin-xx tyramide or DSB-X biotin tyramide, endogenous biotinylated proteins are a potential problem (Figure 12.30). Nonspecific binding of labeled hybridization probes, antibodies and other targeting probes is a potential problem because of the significant signal-amplification capability of TSA. However, because of the high sensitivity of TSA, antibodies and nucleic acid probes can be diluted to far below the amount required for target saturation, thus reducing nonspecific background. Dilution can also substantially reduce the cost of an assay and the amount of an expensive or rare material required for staining. 21 Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes produce substantial background signals when biotin streptavidin detection systems are used to identify cellular targets 75 (Figure 12.29). Because the TSA technology is so sensitive, we recommended preblocking endogenous biotin in cells using our Endogenous Biotin-Blocking Kit (E-21390, Section 7.6) when using our TSA Kits that contain biotin-xx tyramide and the streptavidin conjugate of HRP. This kit provides streptavidin and biotin solutions in convenient dropper bottles and an easy-to-follow protocol. Sufficient material is provided for approximately one hundred 18 mm 18 mm glass coverslips. Improvement of TSA detection by post-incubation heating has been reported. 31 Addition of viscosity-increasing dextran sulfate, poly(vinyl alcohol), poly(ethylene glycol) or poly(vinyl pyrrolidone) to the medium is reported to decrease diffusion of the phenoxy radical intermediate, resulting in superior localization of the signal. 71,76 It has also been reported that hybridization probes that are directly labeled with HRP are particularly useful for lowering nonspecific binding when working with labeled tyramides. 5,33,63 Endogenous peroxidase can be sufficient to yield labeling at the site of this activity in cells, as in the case of eosinophils. 15 The review by Speel, Hopman and Komminoth gives additional practical suggestions and references. 4 References 1. This product is distributed and sold to the enduser pursuant to a license from PerkinElmer Life Sciences, Inc. for use of its tyramide technology by the end-user for life science research applications in the fields of cytochemistry, flow cytometry, histochemistry and in situ hybridization, but not for diagnostic applications. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the licensed use without the express written authorization of PerkinElmer Life Sciences, Inc. and Molecular Probes, Inc. is strictly prohibited; 2. J Immunol Methods 125, 279 (1989); 3. Cytometry 23, 48 (1996); 4. J Histochem Cytochem 47, 281 (1999); 5. J Histochem Cytochem 45, 375 (1997); 6. US 5,196,306; 7. J Histochem Cytochem 45, 315 (1997); 8. J Histochem Cytochem 40, 1457 (1992); 9. Histochem Cell Biol 114, 447 (2000); 10. J Histochem Cytochem 45, 1455 (1997); 11. 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8 References continued 49. Histochem Cell Biol 110, 431 (1998); 50. Biotechniques 27, 608 (1999); 51. J Virol Methods 67, 103 (1997); 52. J Virol Methods 65, 19 (1997); 53. Genomics 44, 355 (1997); 54. Cell Tissue Res 295, 485 (1999); 55. J Histochem Cytochem 46, 1129 (1998); 56. J Histochem Cytochem 44, 1323 (1996); 57. Neurosci Lett 275, 45 (1999); 58. Eur J Cell Biol 79, 394 (2000); 59. J Neurocytol 27, 731 (1998); 60. Acta Histochem 99, 411 (1997); 61. J Comp Neurol 412, 161 (1999); 62. J Histochem Cytochem 44, 1331 (1996); 63. Cytogenet Cell Genet 75, 258 (1996); 64. Mol Cell Probes 13, 261 (1999); 65. Appl Environ Microbiol 65, 1259 (1999); 66. Appl Environ Microbiol 63, 3268 (1997); 67. Genomics 53, 296 (1998); 68. Reg Peptides 80, 67 (1999); 69. Histochem Cell Biol 111, 435 (1999); 70. Neuropathol Appl Neurobiol 22, 548 (1996); 71. J Histochem Cytochem 44, 389 (1996); 72. J Pathol 194, 130 (2001); 73. Arch Pathol Lab Med 102, 113 (1978); 74. J Histochem Cytochem 36, 317 (1988); 75. J Histochem Cytochem 45, 1053 (1997); 76. Histochem Cell Biol 111, 89 (1999). Product List 6.2 Tyramide Signal Amplification (TSA) Technology Tyramide Signal Amplifiction Kits are listed in Table Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology When detecting specific biomolecules in cells, tissues and microarrays, enzyme-mediated detection methods can provide significant signal amplification due to the catalytic turnover of the fluorogenic or chromogenic substrate. For example, NBT (N-6495, Section 10.3) is frequently combined with the phosphatase substrate BCIP (B-6492, Section 10.3) to produce a colored precipitate at the site of phosphatase activity in histochemical assays, in situ hybridization techniques and Western, Northern and Southern blot analyses. Because fluorometric methods are potentially much more sensitive than colorimetric methods, Molecular Probes has been actively engaged in research to develop fluorogenic substrates for enzyme-mediated detection. We now have available reagents and kits to support two outstanding and complementary detection technologies Enzyme-Labeled Fluorescence (ELF) and tyramide signal amplification (TSA, Section 6.2) that provide significantly enhanced detection of targets and unusually high photostability at the same time. Used sequentially, the TSA and ELF technologies should permit ultrasensitive detection of very low abundance targets in cells and tissues that cannot be visualized with any other fluorescent technology. 1 Our patented ELF 97 phosphate is a substrate for both alkaline phosphatase and acid phosphatase with several unique properties that make it superior to many of the existing reagents for detecting these enzymes. 2,3 Upon enzymatic cleavage (Figure 6.16), this weakly blue-fluorescent substrate yields a bright yellow-green fluorescent precipitate that exhibits an unusually large Stokes shift and excellent photostability. The ELF 97 phosphatase substrate is a particularly powerful tool for immunohistochemistry, mrna in situ hybridization methods, 4 6 and detection of DNA on DNA chips. Unlike the radioactive signal produced by conventional methods, ELF 97 mrna detection signals can be developed in minutes or even seconds and can be clearly distinguished from sample pigmentation, which often obscures both radioactive and colorimetric signals. Moreover, in in situ hybridization applications, the yellow-green fluorescent precipitate of the ELF 97 alcohol produces a signal that is many-fold brighter than that achieved when using either directly labeled fluorescent hybridization probes or fluorescent secondary detection methods. 7 9 Spectral Characteristics of the ELF 97 Signal Figure 6.16 shows conversion of the water-soluble ELF 97 phosphatase substrate (E-6588, E-6589) into its hydrolysis product, the ELF 97 alcohol (E-6578), by action of a phosphatase enzyme. Under our reaction conditions, this highly insoluble planar molecule forms an intense yellow-green fluorescent precipitate at the site of phosphatase activity. Like other crystalline fluorescent molecules containing an intramolecular hydrogen bond, the precipitate of the ELF 97 alcohol provides a fluorescent signal that is not only extremely photostable but also has an exceptionally large Stokes shift The ELF 97 signal is ordersof-magnitude more photostable than is possible using either direct or indirect detection with fluorescein conjugates 2,13 (Figure 6.17). This high photostability makes it very easy to focus and photograph ELF 97 alcohol labeled tissue under high magnification. For example, in tissue samples stained using reagents in the ELF 97 Immunohistochemistry Kit, we have observed that about 40% of the ELF 97 alcohol precipitate signal remains following two hours of constant full-power illumination from an epifluorescence microscope. Also, because the fluorescence emission of the Figure 6.16 Principle of enzyme-mediated formation of the fluorescent ELF 97 alcohol precipitate from the ELF 97 phosphatase substrate. Section