Chapter 6 — Ultrasensitive Detection Technology
- Definitions: Detection Reagents
- Primary Detection Reagents
- Secondary Detection Reagents
- Common Experimental Protocols for Primary and Secondary Detection Reagents
- Principles of Tyramide Signal Amplification
- A Variety of Kits for TSA Detection
- TSA Kits
- Zenon Horseradish Peroxidase Antibody Labeling Kits
- Zenon Antibody Labeling Kits Enhanced with TSA Technology
- Applying TSA Technology to Cells and Tissues
- Immunohistochemical Detection Using TSA
- Fluorescence In Situ Hybridization Using TSA
- Detection of Biotin-XX Tyramide, DSB-X Biotin Tyramide and Hapten-Labeled Tyramides
- Double and Sequential Amplification with TSA
- DAB Histochemistry Kits
- Additional Tips on Using TSA Technology
- Our Bibliography of TSA Applications
- Product List
- Spectral Characteristics of the ELF 97 Signal
- Applications of ELF 97 Staining in Histochemistry
- Overcoming Sample Autofluorescence
- ELF 97 Kits for a Wide Variety of Applications
- ELF 97 mRNA In Situ Hybridization Kits
- ELF 97 Cytological Labeling Kit
- ELF 97 Immunohistochemistry Kit
- Endogenous Biotin-Blocking Kit
- ELF 97 Endogenous Phosphatase Detection Kit
- Reagents and Accessories for the ELF 97 Kits
- ELF 39 Phosphate, ELF 97 Glucuronide and ELF 97 Glucosaminide
- ELF Spin Filters
- Data Table
- Product List
- Phycobiliproteins
- Spectral Characteristics of Phycobiliproteins
- B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin
- Tandem Conjugates of Phycobiliproteins
- Pure Phycobiliproteins
- Phycobiliprotein Conjugates
- Reactive Phycobiliprotein Derivative
- Phycobiliprotein-Labeled Secondary Detection Reagents
- Secondary Detection Reagents Labeled with Alexa Fluor Dye–Phycobiliprotein Tandem Conjugates
- R-Phycoerythrin Anti-Fluorescein/Oregon Green Antibody
- Phycobiliprotein Conjugates of Anti-CD Antibodies
- Phycobiliprotein Conjugates of Annexin V
- Custom Phycobiliprotein Conjugates
- Zenon Antibody Labeling Technology
- Product List
- Properties of Our Fluorescent and Nonfluorescent Microspheres
- Fluorescent FluoSpheres and TransFluoSpheres Microspheres
- Colored and Unstained Microspheres
- Applications for Fluorescent Microspheres
- FluoSpheres Fluorescent Microspheres
- A Wide Array of Fluorescent Colors
- A Wide Range of Sizes
- Four Different Surface Functional Groups
- Fluorescent Microspheres Conjugated to Biotin, Avidin and Streptavidin
- Fluorescent Microspheres Coated with Collagen
- Europium and Platinum Luminescent Microspheres for Time-Resolved Fluorometry
- Fluorescent Microsphere Starter Kits
- Fluorescent Microspheres for Educational Purposes
- TransFluoSpheres Fluorescent Microspheres: Tools for Multicolor Detection
- Advantages of TransFluoSpheres Fluorescent Microspheres
- TransFluoSpheres Beads to Match Different Excitation Sources
- BlockAid Blocking Solution
- Our Microsphere Bibliography
- Product List
List of Tables
Table 6.1 — Tyramide Signal Amplification (TSA) Kits
Table 6.2 — Spectral data for B-PE, R-PE and APC
Table 6.3 — Tandem conjugates of R-phycoerythrin (R-PE)
Table 6.4 — Tandem conjugates of allophycocyanin (APC)
Table 6.5 — Molecular Probes' yellow-green–fluorescent FluoSpheres beads compared with other commercially available yellow-green–fluorescent microspheres
Table 6.6 — Fluorescein equivalents in our yellow-green–fluorescent FluoSpheres beads
Table 6.7 — Summary of Molecular Probes' FluoSpheres fluorescent microspheres
Table 6.8 — Summary of biotin-, streptavidin- and NeutrAvidin biotin-binding protein–labeled FluoSpheres microspheres
Table 6.9 — Summary of Molecular Probes' TransFluoSpheres fluorescent microspheres
List of Figures
Figure 6.1 — Simultaneous detection of three gene targets in a whole-mount Drosophila embryo by fluorescence in situ hybridization.
Figure 6.2 — Zebrafish retina. ELF(R) 97 Immunohistochemistry Kit, tetramethylrhodamine wheat germ agglutinin and Hoechst 33342
Figure 6.3 — Luminescent Constellation™ microspheres for imaging.
Figure 6.4 — Schematic diagram of primary and secondary detection reagents
Figure 6.5 — Schematic representation of TSA detection methods applied to immunolabeling of an antigen
Figure 6.6 — Coupling of Alexa Fluor 488 tyramide to protein tyrosine side chains via peroxidase-mediated formation of an O,O'-dityrosine adduct
Figure 6.7 — HeLa cells detected with Alexa Fluor 546 tyramide
Figure 6.8 — Tyramide signal amplification of immunofluorescent staining in mouse brain sections.
Figure 6.9 — Nuclear and nonnuclear incorporation of 5-bromo-2'-deoxyuridine in live cells.
Figure 6.10 — Detection of epidermal growth factor (EGF) receptors directly or with signal amplification
Figure 6.11 — Enhancement of estrogen receptor detection sensitivity by tyramide signal amplification
Figure 6.12 — Zebrafish retina. TSA Kit #2, Alexa Fluor(R) 350 wheat germ agglutinin conjugate and TOTO(R)-3 nucleic acid stain.
Figure 6.13 — In situ hybridization of α-satellite probes to human chromosomes 1, 15 and 17 detected by tyramide signal amplification.
Figure 6.14 — Digital image analysis comparison of in situ–hybridized biotinylated alpha-satellite probes
Figure 6.15 — Principle of the enzyme-mediated formation of the ELF 97 alcohol precipitate
Figure 6.16 — Photostability comparison for tubulin preparations labeled with ELF 97 alcohol or fluorescein
Figure 6.17 — Fluorescence excitation and emission spectra of the ELF 97 alcohol precipitate
Figure 6.18 — ELF 97 photostability under intense UV illumination with a confocal laser-scanning microscope
Figure 6.19 — HeLa cell nuclei. Texas Red(R)-X streptavidin, biotin-XX goat anti–mouse IgG antibody, ELF(R) 97 Cytological Labeling Kit and Hoechst 33258
Figure 6.20 — Osteoblast cells in adult zebrafish head cryosection. ELF(R) 97 Endogenous Phosphatase Detection Kit, Texas Red(R)-X wheat germ agglutinin and Hoechst 33342 nucleic acid stain.
Figure 6.21 — Schematic diagram of the methods employed in our ELF 97 Kits
Figure 6.22 — Prostate carcinoma. ELF(R) 97 mRNA In Situ Hybridization Kit
Figure 6.23 — Mouse fibroblasts. Paclitaxel, biotin-XX goat anti–mouse IgG antibody and ELF(R) 97 Cytological Labeling Kit
Figure 6.24 — Bovine pulmonary artery endothelial cells (BPAEC). Paclitaxel, biotin-XX goat anti–mouse IgG and ELF(R) 97 Cytological Labeling Kit
Figure 6.25 — Bovine pulmonary artery endothelial cells. Biotin-XX phalloidin, ELF(R) Cytological Labeling Kit
Figure 6.26 — Cellular targets developed for visualization with the reagents in our ELF 97 Cytological Labeling Kits
Figure 6.27 — Zebrafish retina. ELF(R) 97 Immunohistochemistry Kit, Hoechst 33342 and tetramethylrhodamine wheat germ agglutinin.
Figure 6.28 — Adult zebrafish intestine. ELF(R) 97 Endogenous Phosphatase Detection Kit.
Figure 6.29 — Endogenous alkaline phosphatase activity of osteosarcoma cells. ELF(R) 97 Endogenous Phosphatase Detection Kit and Hoechst 33342.
Figure 6.30 — Adult zebrafish kidney. ELF(R) 97 Endogenous Phosphatase Detection Kit and propidium iodide.
Figure 6.31 — A comparison of the photobleaching rates of APC and Cy5 conjugates
Figure 6.32 — Absorption spectra for B-PE, R-PE and APC
Figure 6.33 — Emission spectra for B-PE, R-PE and APC
Figure 6.34 — Fluorescence emission spectra of Alexa Fluor dye–conjugates of R-phycoerythrin
Figure 6.35 — Simultaneous detection of three cell surface markers using an Alexa Fluor 610–R-phycoerythrin tandem conjugate, Alexa Fluor 488 dye and R-phycoerythrin labels
Figure 6.36 — Simultaneous detection of three cell surface markers using an Alexa Fluor 647–R-phycoerythrin tandem conjugate, Alexa Fluor 488 dye and R-phycoerythrin labels
Figure 6.37 — Fluorescence emission spectra of allophycocyanin and long-wavelength Alexa Fluor dye conjugates of allophycocyanin
Figure 6.38 — Fluorescence emission spectra of Alexa Fluor 647–R-phycoerythrin streptavidin and Cy5–R-phycoerythrin streptavidin tandem conjugates
Figure 6.39 — Emission spectra of Alexa Fluor 610–R-phycoerythrin and Texas Red–R-phycoerythrin tandem conjugates
Figure 6.40 — Comparison of immunofluorescent staining by R-phycoerythrin–dye tandem conjugates
Figure 6.41 — Analytical size-exclusion chromatograms of free streptavidin and streptavidin, R-phycoerythrin conjugate
Figure 6.42 — R-phycoerythrin used to detect DNA on a microarray
Figure 6.43 — FluoSpheres(R) fluorescent microspheres.
Figure 6.44 — Positively charged nylon membrane. TransFluoSpheres(R) fluorescent microspheres.
Figure 6.45 — Emission spectra of FluoSpheres beads
Figure 6.46 — PS-Speck™ microsphere used to demonstrate a point-spread function of a microscope's optics.
Figure 6.47 — Fluorescence excitation and emission maxima of the FluoSpheres europium luminescent microspheres
Figure 6.48 — Luminescence excitation and emission spectra of the FluoSpheres platinum luminescent microspheres
Figure 6.49 — Schematic diagram of the large Stokes shifts exhibited by our TransFluoSpheres beads
Figure 6.50 — Fluorescence emission spectra of our 488 nm light–excitable TransFluoSpheres beads
List of Technical Notes and Product Highlights
Note 6.1 — Product Highlight: Combining ELF 97 Staining with Other Fluorophores
Note 6.2 — Technical Focus: Limitations of Low Molecular Weight Dyes
Product Highlights
