RAPID COMMUNICATION |
Correspondence to: Kevin A. Roth, Div. of Neuropathology, U. of Alabama at Birmingham (UAB), Sparks Center 961E, 1530 Third Avenue South, Birmingham, AL 35294-0017. E-mail: kroth@path.uab.edu
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Summary |
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Conventional immunofluorescence detection of biologically relevant proteins and antigens in tissue sections is often limited by relatively weak signals that fade rapidly on illumination. We have developed an immunohistochemical protocol that combines the sensitivity of tyramide signal amplification with the photostability of quantum dots to overcome these limitations. This simple method provides a sensitive and stable fluorescence immunohistochemical alternative to standard chromogen detection.
(J Histochem Cytochem 51:981987, 2003)
Key Words: immunohistochemistry, photobleaching, tyramide signal amplification, nanocrystals
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Introduction |
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IMMUNOFLUORESCENCE MICROSCOPY is a well-characterized technique for the detection of proteins and antigens in tissue sections. However, currently available organic fluorescent labels, such as fluorescein, cyanine dyes, and AlexaFluor dyes, are variably limited by relatively rapid and irreversible photobleaching under high-intensity illumination. Although specific photostabilizing buffers have been developed to decrease photobleaching, these buffers may decrease initial fluorescence signal intensity, are variably effective, and are incompatible with some organic fluorophores (
Some of the limitations in immunofluorescent labeling have been overcome by using enzyme-amplification techniques, particularly tyramide signal amplification (TSA; PerkinElmer Life Sciences, Boston, MA), which may dramatically increase immunofluorescence sensitivity over traditional fluorescence detection procedures (
Quantum dots are newly available photostable fluorophores that also overcome some of the limitations of organic fluorescent dyes. These nanocrystals (commercially available as Qdots from Quantum Dot Corporation; Hayward, CA) consist of a semiconductor core of cadmium selenide coated with a shell of zinc sulfide. An additional polymer layer over the quantum dot enhances water solubility and enables conjugation to streptavidin or other biomolecules such as IgG (
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Materials and Methods |
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Materials
Qdotstreptavidin conjugates (Qdot 605, 585, 565, 525) were purchased from Quantum Dot Corporation. Anti-synaptophysin, a mouse monoclonal antibody (MAb), was purchased from Boehringer Mannheim Biochemica (Mannheim, Germany). Anti-MAP2, a mouse MAb, was purchased from Sigma (St Louis, MO). Secondary antibodies (biotinylated and HRP-conjugated anti-mouse antibodies) and Cy3-conjugated streptavidin were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Fluorescein isothiocyanate (FITC)-conjugated streptavidin was obtained from Pharmingen (San Diego, CA). Alexa 546-conjugated streptavidin was purchased from Molecular Probes (Eugene, OR). Avidinbiotin blocking kit was purchased from Vector Laboratories (Burlingame, CA). Biotin tyramide and TSA amplification buffer were obtained from Perkin-Elmer Life Sciences.
Immunohistochemical Methods
Brains were obtained from either adult SpragueDawley rats (Harlan; Indianapolis, IN) or C57BL6/J mice (Jackson Laboratory; Bar Harbor, ME). Embryos were obtained from gestational day 14 C57BL6/J mice. Whole mouse embryos and adult mouse brain tissues were immersion-fixed overnight at 4C in Bouin's fixative. Adult rat brain was immersion-fixed in either Bouin's fixative or 4% paraformaldehyde to compare the effects of the two fixatives on Qdot immunofluorescence detection. Brains used for frozen sections were cryoprotected in 30% sucrose in PBS (10 mM Na2HPO4, 1.4 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.2), frozen in liquid 1,1,1,2-tetra-fluoroethane on dry ice, and then sectioned by cryostat into 10-µm sections. Four-µm-thick paraffin sections were prepared on a microtome.
After rehydration of frozen sections in PBS or deparaffinization and rehydration of paraffin-embedded sections in serial solutions of Citrisolv (Fisher Scientific; Pittsburgh, PA), isopropyl alcohol, and water, selected tissue sections underwent antigen retrieval by steaming in 10 mM citric acid, pH 6.0, for 20 min and cooling for 20 min. The remaining steps were performed at room temperature unless indicated otherwise. Endogenous peroxidase activity was inhibited by incubating paraffin sections in 3% H2O2 in PBS for 5 min or frozen sections in 0.3% H2O2 in PBS for 15 min, followed by three 5-min washes in PBS. Sections were then incubated for 30 min in PBSblocking buffer (PBS-BB; 1% bovine serum albumin, 0.2% powdered skim milk, 0.3% Triton X-100 in PBS) followed by incubation overnight at 4C with primary antibody diluted in PBS-BB or in PBS-BB alone. Slides were then rinsed three times for 5 min in PBS before application of the secondary antibody in PBS-BB for 1 hr (biotinylated and HRP-conjugated anti-mouse antibodies were diluted 1:500 and 1:2000, respectively).
In experiments employing biotinylated secondary antibody, slides were washed three times for 5 min in PBS and incubated with Qdotstreptavidin conjugates for 30 min at the indicated concentration. Unless specified, experiments were performed using Qdot 605streptavidin conjugate that was suspended in the manufacturer-supplied buffer and preincubated for 30 min at RT before application to the sections. In specific experiments, TSA was performed using HRP-conjugated donkey anti-mouse IgG and biotinylated tyramide diluted 5 ug/ml in TSA amplification buffer. After a 10-min TSA reaction, slides were washed three times for 5 min each in PBS and then incubated with fluorophore-conjugated streptavidin, diluted in PBS-BB, for 30 min at RT. Cy3, FITC, and Alexa 546streptavidin conjugates were used at a concentration of 1 µg/ml. Qdot 605 and Qdot 585streptavidin conjugates were used at a 20-nM concentration and were diluted in manufacturer-supplied buffer. Slides were then washed three times for 5 min in PBS and coverslipped in 90% glycerol10% PBS.
Imaging Methods
Immunofluorescence samples were examined with a Zeiss-Axioskop microscope equipped with epifluorescence. Sections labeled with Cy3, Alexa 546, Qdot 605, Qdot 585, or Qdot 565 were viewed with a Chroma filter set consisting of an excitation filter of 545/30 nm, dichroic of 575 nm longpass, and an emission filter of 610/75 nm. Sections labeled with FITC or Qdot 525 were viewed with a Chroma filter set consisting of an excitation filter of 470/70 nm, dichroic of 500 nm longpass, and an emission filter of 535/40 nm. Digital images were captured with a Zeiss Axiocam and Axiovision software using identical acquisition parameters per experiment. Multiple x40 fields were captured from each tissue section. Care was taken to ensure that images were obtained from consistent regions (i.e., the cerebral cortex of adult brains or spinal cord of embryos) and that the peak pixel intensity for a specific experiment did not exceed the range of the arbitrary optical density units. Images were saved as TIF files and immunofluorescence was quantified using Scion Image freeware (Scion; Frederick, MD) by measuring the average pixel intensity for the entire image. Values are the mean ± SEM of three or four fields per tissue section.
Statistics
In the photostability study, the normalized pixel intensities at time 0 were compared to later time points using paired Student's t-test comparisons with Bonferroni correction for multiple comparisons.
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Results |
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Optimization of Qdot Concentration
In preliminary experiments, we determined that use of a primary antibody with strong, diffuse uniform staining enabled optimal quantitation of immunofluorescence. Synaptophysin is an integral membrane component of presynaptic vesicle membranes of differentiated neurons and is widely distributed throughout the nervous system (
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As the Qdot 605 concentration increased, we observed a diffuse "haze" in both antibody- and PBS-BB-incubated sections. This nonspecific signal was observed in both paraformaldehyde- and Bouin's-fixed tissue and in both frozen and paraffin-embedded sections. Interestingly, the haze faded markedly with illumination to barely detectable levels. To limit this confounding "nonspecific" effect, all fields were exposed to 4 min of illumination before capture of images by digital camera. No further reduction of background was seen with additional illumination. Furthermore, no loss of specific Qdot 605 labeling occurred with prolonged light exposure (see below).
In sections incubated with higher concentrations of Qdot 605 (40 nM), Qdot-specific, antibody-independent labeling of neuronal cell bodies was observed. This labeling was present even in the absence of secondary antibody, as rehydrated brain sections treated only with 40 nM Qdot 605 showed similar neuronal labeling (Fig 1C). This Qdot-specific, antibody-independent labeling was also observed to a lesser degree with 40-nM concentrations of Qdots 585, 565, and 525 (data not shown). In contrast to the diffuse background staining, which faded after several minutes of illumination, this Qdot-specific neuronal staining did not diminish with prolonged light exposure. The neuronal labeling did not appear to be related to endogenous biotin in the tissue sections because pre-blocking with avidin and then biotin (Avidin-Biotin Blocking Kit; Vector Laboratories) did not significantly inhibit the Qdot neuronal staining, nor was it observed with organic fluorescent dyes (data not shown). This neuronal staining was observed in both paraffin sections of Bouin's-fixed sections of adult mouse brain and in frozen sections of both paraformaldehyde- and Bouin's-fixed adult rat brain. The Qdot concentration-dependent neuronal staining was more pronounced in tissue sections subjected to antigen retrieval than in standard sections.
Increased Sensitivity with Tyramide Signal Amplification
We next determined whether a TSA reaction with biotinylated tyramide deposition would improve the sensitivity of Qdot detection. Bouin's-fixed frozen rat brain sections were treated with increasing concentrations of mouse anti-synaptophysin antibody (0.1100 ng/ml) followed by incubation with either biotin-conjugated donkey anti-mouse IgG or HRP-conjugated donkey anti-mouse IgG, followed by indirect TSA with biotin tyramide. Both sets of slides were then incubated with Qdot 605streptavidin conjugate (20 nM) for 30 min and were coverslipped in 90% glycerol10% PBS.
TSA produced a dramatic increase in Qdot 605 detection of synaptophysin immunoreactivity compared to that achieved with biotinylated secondary antibody detection (Fig 2). Signal-to-noise ratio was dramatically improved and the antibody concentration required to detect specific synaptophysin signal was markedly reduced. Similar results were observed with other mouse monoclonal and rabbit polyclonal antibodies (results not shown). Even with the increased sensitivity afforded by TSA detection, minimal changes in background staining were observed.
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Photostability of Quantum Dots Compared with Other Fluorophores
One of the reported advantages of Qdot detection is photostability. To confirm this, Bouin's-fixed, paraffin-embedded 14-day gestation mouse embryo sections were antigen-retrieved and incubated with MAP2 antibodies followed by TSA and detection of biotintyramide with the streptavidin conjugates of Cy3 (1 µg/ml), FITC (1 µg/ml), Alexa 546 (1 µg/ml), Qdot 605 (20 nM), or Qdot 585 (20 nM). MAP2 is present at high levels in neuronal cell bodies, axons, and dendrites during early nervous system development (
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Loss of Qdot Signal After Prolonged PBS Incubation
One of the initial limitations of quantum dots in biological applications was the relative water-insolubility of the nanocrystal. This was overcome by coating the core with a polymer that improved water solubility and hence compatibility with both fixed and living cells (
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Discussion |
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Quantum dots are high-intensity photostable fluorophores that have several distinct advantages over standard organic fluorescent dyes. Analogously, TSA immunodetection provides increased sensitivity over standard immunohistochemical (IHC) procedures. Our study demonstrates that the combined application of TSA and Qdots results in a dramatic increase in the sensitivity and photostability of IHC fluorescence detection.
The semiconductor nanocrystals that comprise quantum dots have been studied for several decades, but the hurdles of achieving water solubility and conjugation to biomolecules have been overcome only recently (
Photostability is one of the main advantages of Qdot detection. We found no loss of immunofluorescence intensity in Qdot-labeled samples even after 1 hr of continuous illumination, whereas there was nearly complete loss of FITC and Alexa 546 immunofluorescence after 5 min of illumination, and loss of Cy3 labeling within 20 min. On the basis of the manufacturer's recommendation, all Qdot-labeled slides were coverslipped with 90% glycerol/10% PBS. In preliminary experiments we found that this high concentration of glycerol enabled optimal fluorescence detection compared to 50% glycerol/50% PBS. Although antifade mounting media would have undoubtedly improved the stability of the organic dye fluorophores tested, even with antifade media
We found linear increases in specific immunostaining with increasing Qdot 605 concentration up to 20 nM, whereas nonspecific background staining did not significantly increase until >20 nM Qdot 605 concentrations were used. During the course of these studies we observed several unusual features of Qdot detection. First, neuronal cell body staining was noted at high concentrations of Qdot 605, which was Qdot-specific and antibody-independent. This neuronal staining was most prominent with Qdot 605streptavidin conjugate but was also observed in brain sections incubated with 40 nM Qdot 585, 565, or 525streptavidin conjugates. Even though we routinely use Qdot conjugates at concentrations that do not exhibit this effect, i.e., 20 nM, we now include a control slide in each experiment that is incubated only with the Qdot conjugate to monitor any Qdot-dependent labeling. Nonspecific staining has been described to be a limitation of quantum dots (
Second, we often observed a diffuse background "haze" in our Qdot-labeled sections that faded on illumination. Pre-illumination of the field of interest for several minutes dramatically decreased the haze and improved the signal-to-noise ratio. This diffuse photobleachable background was much less prominent at lower concentrations of Qdot 605, indicating that this background labeling is also a Qdot concentration-dependent effect.
Although streptavidin-conjugated quantum dots have the distinct advantage of photostability, we did observe loss of signal intensity after prolonged incubation of Qdot-labeled sections in aqueous buffer. This is unlikely to be caused by simple diffusion of streptavidin-conjugated label from the tissue because no loss of signal was observed for streptavidinCy3-labeled tissue sections after >24 hr in PBS. Despite modifications to the quantum dot nanocrystal to improve water solubility (
We have also occasionally observed loss of Qdot 605 signal in frozen brain sections coverslipped with 90% glycerol and stored for several weeks, regardless of whether slides were stored at 4C or at RT. Care was taken to ensure that the tissue sections did not dry because preliminary studies demonstrated dramatically decreased Qdot 605 signal if tissue sections had dried after Qdot labeling. No loss of Qdot 605 signal has been observed in immunostained paraffin-embedded tissue sections coverslipped in 90% glycerol and stored for up to 2 months at 4C. Further studies will be required to determine the stability of Qdots under various slide storage conditions.
This is the first description of the combined application of quantum dots and TSA for IHC detection. The increased sensitivity of the TSA reaction enables use of the primary antibody within a linear range of concentrations and, now that a photostable detection method is available, it may be possible to accurately quantitate antigen levels in tissue sections using the combination of TSA, Qdot detection, and immunofluorescence microscopy.
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Acknowledgments |
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Supported by the Dixon Foundation and the Research Institute of the Children's Hospital of Alabama (JMN), and by a grant from the University of Alabama at Birmingham (UAB) Health Services FoundationGeneral Endowment Fund (KAR). RSA received support from the UAB Medical Scientist Training Program (TM GM0831).
We thank Ms Angela Schmeckebier and Ms Jo Self for assistance in the preparation of this manuscript.
Received for publication May 5, 2003; accepted May 14, 2003.
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