TECHNICAL NOTE |
Correspondence to: John T. Povlishock, Dept. of Anatomy, Medical College of Virginia, Virginia Commonwealth U., Richmond, VA 23298-0709.
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Summary |
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Fluorescent immunocytochemistry (FICC) allows multiple labeling approaches when enzyme-based techniques are difficult to combine, such as in double-labeling experiments targeting small-caliber axonal segments. Nevertheless, the conversion of FICC to a product visible at the electron microscopic (EM) level requires labor-intensive procedures, thus justifying the development of more user-friendly conversion methods. This study was initiated to simplify the conversion of FICC to EM by employing the unique properties of tyramide signal amplification (TSA), which allowed the simultaneous targeting of a fluorescent tag and biotin label to the same antigenic site. Briefly, one of two antigenic sites typically co-localized in damaged axonal segments was visualized by the application of a fluorescent secondary antibody, with the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophoretyramide and biotintyramide. After temporary mounting for fluorescent digital photomicroscopy, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clearly delineated damaged axonal segments. In addition, these same axonal segments yielded high-quality EM images with discrete electron-dense reaction products, thereby providing a simple and reproducible means for following fluorescent analysis with EM. (J Histochem Cytochem 48:153161, 2000)
Key Words: tyramide signal amplification, axonal injury, immunofluorescence, double labeling, trauma, calpain
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Introduction |
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THE COMBINATION of fluorescent tags with distinct emission spectra and the use of enzyme-based staining procedures constitute the most widely used tools in double/multiple-labeling immunocytochemistry (ICC). Although enzyme-based multiple-labeling techniques are excellent for the analysis of axodendritic and axosomatic reactions or for immunocytochemical co-localization of nuclear and cytoplasmic antigens, the spatial resolution of these techniques is generally insufficient to detect antigens co-localized in the same axonal segment.
Fluorescent markers are more appropriate for such studies. However, their use necessitates further conversion methods when subsequent electron microscopic (EM) investigations are needed to confirm their precise sites of localization (
Recently, in our laboratory we have successfully utilized a combination of the peroxidase-based chromogens benzidine dihydrochloride (BDHC) and diaminobenzidine (DAB) or Vector VIP for both LM and EM co-localization of reaction products linked to calcium-induced, calpain-mediated spectrin proteolysis (CMSP) and cytoskeletal change in traumatically injured axonal segments (
Recognizing that the TSA technique allows the simultaneous application of a fluorescent tag [fluorophore (rhodamine)tyramide] and biotin (biotintyramide) to the same antigenic site, we describe here a substantial modification of the conventional TSA technique that yields a reliable and simple method that can be employed for fluorescent double/multiple-labeling experiments combined with subsequent pre-embedding immunoelectron microscopic analysis of the labeled structures. We also comment on the utility of this method in the study of axonal profiles, in which it combines the advantages of sensitive signal amplification with a user-friendly conversion method for immunoelectron microscopy.
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Materials and Methods |
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Induction of Traumatic Brain Injury
For induction of focal axonal injury, a well-characterized rodent model of impact acceleration head injury was employed as described by
Physiological Assessments
The respiratory status was monitored through pulse oximetry via the footpad and/or the tongue. In addition, brain temperature was monitored by a temporalis muscle probe and core temperature was determined by a rectal probe.
Immunohistochemistry
At the designated survival time, the rats were reanesthetized with an overdose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in Millonig's buffer. The brains were immersed in the same fixative overnight (1618 hr). On the basis of previously published observations concerning the topography of diffusely injured axons in rat brain (-subunit of brain spectrin on its cleavage by the calcium-activated protease calpain, allowing the identification of the precise locus of any calcium-mediated events linked to the proteolysis of spectrin at both the LM and EM level (
After rinses in 1% NDS/NGS, sections were incubated in a solution of coumarin-labeled donkey anti-mouse immunoglobulin (1:200; Jackson Immunoresearch Laboratories, West Grove, PA) and biotinylated goat anti-rabbit immunoglobulin (1:400; Vector, Burlingame, CA) for 60 min, followed by three 10-min rinses in PBS. In all subsequent steps of tissue processing, direct illumination of the sections was avoided.
Tyramide Signal Amplification
Based on attempts to simplify the TSA method and make it more cost-effective, while also providing better preservation of ultrastructural detail several modifications of the original protocol (for details see the commercial protocol, Renaissance Kit, NEN Life Sciences Products, Boston, MA) were introduced in our laboratory. Specifically, after incubation in an avidinbiotinperoxidase complex (ABC standard Elite kit; Vector; dilution 1:200) with rinsing in TNB blocking buffer (Renaissance Kit, NEN Life Science) for 20 min and in PBS twice for 10 min, a 1:1 mixture of rhodaminetyramide and biotintyramide (Renaissance Kit, NEN Life Science), both diluted 1:300, was applied. A 1:6 mixture of amplification diluent (Renaissance Kit, NEN Life Science) and PBS constituted the solvent for the tyramide stock solution, together forming the working solution. The working solution was applied in mid-sized tissue culture wells containing six tissue sections each (12-well/tray; Falcon Multiwell Tissue Culture Plate, Becton Dickinson Labware; Becton Dickinson, Lincoln Park, NJ), 400 µl working solution/well, for 12 min, followed by three 10-min rinses in PBS. The dilution of the amplification diluent in PBS to 1:6, as well as the substitution of the detergent-containing rinsing buffer (Tween is suggested in the original protocol) with PBS was aimed to prevent unnecessary membrane damage and reduce the background reactivity. Next, the sections were mounted on premium microscope slides (Fisher Scientific; Pittsburgh, PA) and coverslipped using 50% glycerol dissolved in double-distilled water. The slides were immediately transferred to a Nikon Eclipse E 800 biological research microscope (Image Systems; Columbia, MD) equipped with a Sony Catseye digital camera (Image Systems). Images of immunofluorescent axonal profiles were digitally captured and archived. All the sections analyzed were mounted and coversliped separately, with each assigned a serial ID number. After completion of digital acquisition, coverslips were removed and the sections were re-rinsed and incubated in an avidinbiotinperoxidase complex at a dilution of 1:200. The sections were then processed for visualization of the immunohistochemical complex using 0.05% diaminobenzidine (DAB) (Sigma) and 0.01% H2O2 in 0.1 M phosphate buffer. Although in theory one could argue that the previously applied peroxidase molecule found in the ABC complex, used to activate tyramide, could also serve as the catalyst for the chromogenic reaction with DAB, this approach consistently yielded such a weak signal as to preclude its usefulness for routine LM/EM studies.
Ultrastructural Analysis
After the above prepared sections were dehydrated and flat-embedded between plastic slides in Medcast resin (Ted Pella), areas for EM investigation were selected by repeated, detailed analysis involving comparisons with the previously captured digital images. To draw a precise correlation between these samples assessed either with fluorescence or routine light microscopy, we employed anatomic landmarks such as the vicinity of blood vessels, tissue edges, and the relative distance between labeled axonal segments. Once identified, homologous immunopositive foci prepared for ultrastructural analysis were trimmed, mounted on plastic studs, and sectioned using an LKB Ultratome at a thickness of 70100 nm. A few semithin sections were mounted and coverslipped for comparison to the digital images while also serving as topographic guides for the ultrastructural analysis. Thin sections were picked up on Formvar-coated slotted grids, then stained in 5% uranyl acetate in 50% methanol for 2 min and 0.5% lead citrate for 1 min. Alternatively, some sections were not stained to provide additional evidence for antibody specificity. Ultrastructural analysis was carried out using a JEOL-1200 electron microscope.
Immunohistochemical Controls
The antibodies employed have been extensively characterized in the existing literature and widely used (
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Results |
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Fluorescent and Routine LM Observations
In accord with the results of our previously published studies, traumatically injured axonal profiles displayed focal axonal immunofluorescence representing neurofilament compaction (RMO-14) and calpain-mediated spectrin proteolysis (Ab38), with the highest proportion of these profiles found in the corticospinal tract and in the medial longitudinal fasciculi (
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Ultrastructural Observations
In this approach, the overall ultrastructural appearance of the tissue described above was well-preserved despite these additional tissue-processing procedures. At the EM level, damaged immunoreactive axons displayed the well-established repertoire of traumatic axonal injury, including neurofilament compaction, mitochondrial swelling, loosening of the myelin sheath, and formation of periaxolemmal spaces (
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Immunohistochemical Controls
The exclusion of either the primary or the secondary antibody from the immunohistochemical reaction resulted in lack of immunoreactivity. Gradual overdilution of the immunosera caused disappearance of the immunostaining. Simultaneous or sequential application of immunosera in the case of double labeling F-IHC resulted in equally powerful detection of both antigens. The TSA kit alone did not contribute to any specific immunohistochemical reaction that might have influenced the analysis. Elimination of the rhodaminetyramide step resulted in the lack of fluorescent labeling of CMSP-IR axonal profiles, while the absence of biotintyramide resulted in the lack of specific DAB staining (i.e., lack of conversion) at the LM level without affecting the fluorescence signal (rhodamine label) indicating CMSP-IR.
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Discussion |
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In a series of labor-intensive studies employing the combination of enzyme-based immunohistochemical reaction products (DAB/VVIP and BDHC/VVIP), we have recently demonstrated the co-localization of calpain-mediated spectrin proteolysis (CMSP) with markers of traumatically induced cytoskeletal alteration, providing evidence for the contribution of CMSP to the pathogenesis of traumatically induced axonal injury (TAI) (
In the present study, the combination of the conventional immunofluorescence (coumarin-labeled marker for neurofilament compaction) and fluorophore-labeled tyramide (rhodamine marker for calpain-mediated spectrin proteolysis) yielded high-quality fluorescent labeling that consistently denoted sites of injury previously described in this model of TAI. Further, the conversion of the tyramide label to an electron-dense DAB reaction product allowed this same fluorescently tagged structure to be easily carried forward to the EM level. We emphasize that the reaction presented here does not involve a direct conversion of the rhodamine tag but rather proceeds in relation to the co-localized biotintyramide. This strategy provided extremely high resolution and fidelity because virtually all the axonal profiles detected with the rhodamine tag were also labeled with DAB. It should be noted that none of the axonal profiles displaying single labeling with the coumarin marker alone demonstrated DAB reactivity. This finding further underlines the specificity and fidelity of the technique. As noted, at the ultrastructural level the tissue was well preserved, with the suggestion that the intensity of the staining was even more pronounced than that achieved through the tradititional HRPDAB methods. Although we are uncertain as to why this was the case, we postulate that the concomitant presence of the electron-dense tyramide molecule itself enhanced the density of the overall reaction product (
In relation to the observed electron-dense reaction, we also note that the reaction product appeared to be of consistent size and density, showing consistent localization. To be candid, we were surprised by this finding because the histochemistry underlying tyramide amplification involves a tyramidetyrosine covalent binding that is not directly determined by immunological principles (
Why our method is not confounded by this liability is unknown. However, we speculate that our modification of the original commercially available TSA protocol at several key steps may have provided optimal signal-to-noise ratio at both the LM and EM level while achieving an optimal costbenefit ratio.
Also of note in this process is the fact that the simultaneous visualization of the biotin molecule by the DAB chromogen enables the investigator to overcome the problem of fluorescent signal fading over time.
We believe that our study convincingly demonstrates the versatility of the TSA method. Therefore, in addition to being an excellent technique for augmenting immunocytochemical reactions, it is also considered an outstanding tool for immunofluorescent double/multiple labeling, with the possibility of a simple signal conversion method for EM. Furthermore, because the simultaneous application of biotin and rhodaminetyramide facilitates ultrastructural analysis of double-labeled immunofluorescent profiles, this opens up new potential for the future use of this sensitive, versatile signal amplification technique.
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Acknowledgments |
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Supported by grants NS 20193 and by the Martin Rodbell Fellowship.
We thank Dr Robert Siman for kindly donating the Ab38 and Dr John Q. Trojanowski for the RMO-14 antibody. We also thank Lynn Davis, Thomas Coburn, and Judy Williamson for excellent technical support.
Received for publication June 22, 1999; accepted August 31, 1999.
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