TECHNICAL NOTE |
Correspondence to: Mario I. Romero, Dept. of Anesthesiology and Pain Management, U. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235.
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Summary |
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Visualization of the neuronal tract tracer horseradish peroxidase (HRP) is commonly achieved through the histochemical detection of its enzymatic activity using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. However, the TMB product is unstable and is incompatible with tissue processing methods that render the enzyme inactive, or when a combination of HRP tract tracing with neuronal phenotype identification is required. In this study we evaluated the applicability of the immunocytochemical detection method for horseradish peroxidase (HRP) visualization using an enhanced detection meth-od based on the Elite ABC peroxidase amplification protocol. The results provide evidence for the immunocytochemical visualization of both anterograde and transganglionic HRP transport in the rat spinal cord. This immunocytochemical method not only showed similar sensitivity to the TMB protocol in detecting HRP-labeled motor neuron perikarya but provided enhanced resolution in the identification of individual neuronal fibers compared to the TMB method. Immunodetection of the HRP tracer also allowed its co-localization with specific neuronal markers using double immunofluorescence techniques. These results offer the first demonstration that sensitive identification of axonally transported HRP can be achieved by immunocytochemistry and provides further support for its use in HRP tract tracing studies. (J Histochem Cytochem 47:265272, 1999)
Key Words: neuroanatomic tract tracing methods, horseradish peroxidase, tetramethylbenzidine, Elite ABC, immunocytochemistry, double labeling, spinal cord
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Introduction |
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The enzyme horseradish peroxidase (HRP), either unmodified or lectin-conjugated, is widely used as a reliable and sensitive marker for tracing neural pathways in the nervous system (
Immunocytochemical localization of HRP was recognized early as an alternative method for HRP visualization, particularly useful in cases in which the enzymatic activity of HRP was lost during tissue processing (
In this study we evaluated the use of an enhanced amplification method for immunocytochemical detection of HRP based on the Elite avidinbiotinperoxidase complex (Elite ABC; Vector Laboratories, Burlingame, CA). The results indicate that visualization of both anterograde- and transganglionic-transported HRP with the Elite ABC immunocytochemical detection protocol provides similar sensitivity and enhanced resolution compared to the TMB method. Furthermore, exclusion of glutaraldehyde in the fixative solution allowed the use of double immunofluorescence in the detection of tracerHRP and specific cell phenotype antigens.
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Materials and Methods |
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Animal Surgery
Experiments were performed on adult (250350 g) female SpragueDawley rats (Harlan Sprague Dawley; Harlan, TX). The animals were anesthetized with an IP injection of a ketamine (67 mg/kg)/xylazine (6.7 mg/kg) solution before the application of the HRP tracers for both transganglionic and retrograde tract tracing mapping, as described below. After completion of the injections, the muscle layers were sutured, clips were applied to the skin incision, and prophylactic antibiotic treatment was applied topically to the wound. Animals were observed continually after the surgery, and normal body temperature was supported until recovery from anesthesia. The animals were maintained under conditions of controlled light and temperature, and food and water were available ad libitum. Institutional Animal Care and Research Advisory Committee regulations were observed for surgical and care procedures.
Transganglionic Tract Tracing
Five animals were used for transganglionic HRP labeling. The right sciatic nerve was surgically exposed at the mid-thigh level in these animals and a strip of parafilm was inserted temporarily under the nerve to prevent leakage of the tracer into the surrounding tissue. Using a Nanoject injector (Drummond Scientific; Broomall, PA.)-driven glass micropipette, 47 µl of an HRP mixture (20% HRP Type IV; Sigma Chemical, St Louis, MO/5% WGAHRP; Sigma Chemical/0.2% B-choleratoxinHRP; List Biological Labs, Campbell, CA/5% dimethylsulfoxide) was directly injected into the nerve 0.5 cm distal from its emergence from the great sciatic notch. The micropipette was left in place for 5 min after injection to allow diffusion of the tracer before closing the wound. B-choleratoxin HRP was omitted from the HRP mixture in two animals to allow the specific visualization of unmyelinated C-fibers in the lamina II of the dorsal horn of the spinal cord (
Anterograde Tract Tracing
Hemilaminectomies were performed in three separate animals at the T13L2 vertebral segments to expose the L4L5 dorsal roots. The isolated roots were placed on a parafilm strip and transected by crushing the roots twice for 10 sec each, using 0.5-mm jewelers' forceps, taking care not to disrupt the perineurium. The peripheral end of the root was ligated with 6-0 suture to facilitate the preferential diffusion of the tracer towards the spinal cord. Using a Nanoinjector-driven micropipette, 4 µl of the HRP mixture was delivered over a 2-min period directly into the lesioned area of each dorsal root. The micropipette was left in place for 2 min after injection to allow tracer diffusion.
Histochemistry and Immunocytochemistry
At the end of the treatment period (i.e., 24 hr for anterograde labeling and 48 hr for transganglionic tract tracing), the animals were anesthetized and perfused transcardially with either 1% paraformaldehyde/1.5% glutaraldehyde or 4% paraformaldehyde in buffered saline. The lumbar spinal cord was then removed and postfixed for at least 24 hr at 4C. Coronal tissue sections were obtained either at 10 µm using a cryostat (Hacker-Bright Instrument; Huntingdon, UK) or 50 µm using a Vibratome (Lancer; Technical Products International, St Louis, MO). The tissue sections were divided into five alternate sets at 250-µm and 50-µm intervals for microtome and cryostat sections, respectively, and were either processed immediately or stored in cryoprotectant solution (
The TMB Method
Visualization of HRP was performed according to the protocol described by
Anti-HRP Immunocytochemistry
After removal of residual fixative from the tissue sections by extensive PBS, pH 7.5, rinses, the sections were incubated in 3% hydrogen peroxidase to quench endogenous peroxidase activity. After further rinsing, the tissue was then incubated with 5% normal goat serum (NGS) to reduce nonspecific staining and subsequently incubated in a rabbit polyclonal HRP antiserum (1:20,000; Sigma) solution for 24 hr at RT with continuous agitation. Visualization was achieved by tissue incubation in biotinylated goat anti-rabbit IgG secondary antibodies (1:600). Biotin-labeled tissue was further processed using the Vectastain Elite ABC reagents and was developed with a solution of hydrogen peroxide (0.003%) and diaminobenzidine (0.02%). The specificity of the HRP antiserum was corroborated by the lack of staining in tissue in which the primary antibody was excluded from the staining protocol. In addition, the localization of transported HRP was completely abolished when the HRP antiserum was preabsorbed with HRP (1 x 10-6 M, Type IV; Sigma) 24 hr before its use for immunocytochemistry. Tissue sections of both control and experimental groups were simultaneously developed in identical incubation solutions. Sections were mounted on gelatinized slides and coverslipped for microscopic evaluation.
Double Immunofluorescence
After incubation of the tissue with 5% NGS, the sections were incubated simultaneously with the rabbit anti-HRP (1:4000) and monoclonal antiserum for the nonphosphorylated form of neurofilament H (MSI-32; 1:500; Sternberger Monoclonals, Lutherville, MA). Visualization of the primary antibodies was achieved by simultaneous incubation in Texas Red-labeled goat anti-rabbit and fluorescein-labeled goat anti-mouse (1:250 for both antibodies; Jackson ImmunoResearch Laboratories, West Grove, PA). The sections were mounted on slides and coverslipped with 5% N-propyl-gallate in glycerol and were visualized by epi-illumination.
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Results |
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Transganglionic Tract Tracing
After injection of an HRP/WGAHRP solution to the sciatic nerve, axonally transported HRP was localized preferentially in lamina II of the dorsal horn (Figure 1A and Figure 1B) and in the motor neurons of the ventral horn (Figure 1CF). Identification of HRP-containing fibers and cell bodies in these areas appeared qualitatively similar with either TMB (Figure 1A, Figure 1C, and Figure 1E) or immunocytochemical (Figure 1B, Figure 1D, and Figure 1F) visualization. Visualization of the HRP by the TMB method, however, also resulted in moderately high background, characterized by nonspecific deposition of TMB crystals over the tissue (Figure 1A and Figure 1C). Nonspecific staining was also observed in blood vessels, indicating the presence of residual erythrocytes. In contrast, immunodetection with the enzyme resulted in very specific staining of the labeled neuronal fibers and cell bodies, with minimal background and no detection of immunoreactivity in other types of cells (Figure 1B, Figure 1D, and Figure 1F). Identification of labeled ventral motor neurons was similarly achieved by the two HRP detection methods, as indicated by the comparable number of stained perikarya (Figure 1C and Figure 1D). Conversely, visualization of individual motor neuron processes, such as axons or dendrites, appeared to differ between the two visualization methods. The granulated nature of the TMB staining yielded discontinuous labeling of axons and dendrites which, along with the highly granular background, made the identification of individually labeled processes a challenging task. This was particularly evident when visualization of individual labeled fibers distal from the neuron perikarya was attempted (Figure 1E). In contrast, the use of anti-HRP antibodies for HRP detection permitted the identification not only of the labeled cell bodies but also of the HRP-containing neuronal processes (Figure 1F), suggesting that the resolution achieved by the immunocytochemical method is greater than that of the TMB protocol.
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Anterograde Tract Tracing
Application of the lectin-conjugated HRP mixture to the dorsal roots resulted in the labeling of sensory axons in the spinal cord as detected by both the TMB (Figure 2A and Figure 2C) and the anti-HRP (Figure 2B and Figure 2D) method. However, the two protocols differed in the qualitative signal-to-noise ratio and consequent resolution in HRP detection. The nonspecific background was higher in the TMB-processed tissue, in which TMB crystals of moderate to large size were found throughout the tissue. Evaluation of the traced axons at higher magnifications further emphasized the difference in signal-to-noise ratio yielded by the TMB and the anti-HRP protocol (Figure 2B and Figure 2D). Where-as the TMB visualization of HRP-labeled axons was granulated, discontinuous, and obscured by nonspecific deposits of TMB crystals (Figure 2B), the immunocytochemically stained fibers were clearly and more specifically defined (Figure 2D). In addition, the numbers of labeled fibers appeared to be increased in sections processed immunocytochemically (compare Figure 2B and Figure 2D).
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Simultaneous Visualization of Axonally Transported HRP and Cell-specific Markers
Figure 3 illustrates the simultaneous visualization of axonally transported HRP (Figure 3A) and the neurofilament neuronal cell marker (Figure 3B) in the ventral horn of the spinal cord by double immunofluorescence. Co-localization of the two markers in the perikarya (arrows) and neurites (arrowheads) of ventral motor neurons demonstrates the ability of the immunocytochemical method for the simultaneous identification of the tracer with specific cell phenotype markers in these cell compartments.
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Discussion |
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Unmodified and lectin-conjugated HRPs are widely used as reliable and sensitive markers for tracing neural pathways (
In this study we have reevaluated the applicability of the immunocytochemical detection method using a more recent and enhanced detection method based on the Vector Elite ABC peroxidase amplification protocol. This method is based on equimolar ratios of avidin D and biotinylated peroxidase (
Detection of Axonally Transported HRP
Our results provide evidence for the immunocytochemical visualization of both anterograde and transganglionic HRP transport in the rat spinal cord. Anterograde transport was observed after injection of the HRP tracer into the primary dorsal afferents and subsequent visualization of the enzyme in the dorsal and ventral horns of the spinal cord. Transganglionic HRP transport was verified by visualization of the HRP tracer in the dorsal root ganglia (not shown) and in both primary sensory afferents, as well as in ventral motor neurons, after injection of the tracer into the sciatic nerve. In all cases evaluated, the sensitivity of the immunocytochemical method for visualization of the HRP tracer was at least qualitatively similar to that obtained by the TMB method. This is clearly indicated by the apparently equal number of ventral motor neurons identified by the two methods.
Enhanced Resolution of HRP Visualization by Immunocytochemistry
Visualization of axonally transported HRP by the TMB method, although sufficient for localization of labeled perikarya, is unsatisfactory for identification and tracing of neuronal fibers. The identification of neurites by TMB is complicated by the granular and discontinuous nature of the TMB precipitation product, which limits the appreciation and tracing of fine dendrites and axons. The nonspecific deposition of TMB crystals on the surface of tissue sections further obscures the detail of the labeled pathways. Conversely, the immunocytochemical method allows detailed identification of neurites and facilitates the tracing of individual neuronal fibers. This observation most likely reflects the differences between the DAB and the TMB chromogens. In contrast to the granular nature of the TMB precipitate, the DAB product is noncrystalline and therefore diffuses freely through the fibers, allowing more even and continuous labeling. In addition, discrimination of individual labeled fibers was facilitated by the low nonspecific staining achieved by the immunodetection protocol. Suppression of endogenous peroxidase activity and the use of low titers of anti-HRP antibodies (i.e., 1:20,000) reduced the nonspecific staining compared to the TMB method, thereby enhancing the signal-to-noise ratio.
Co-localization of the HRP Tracer and Cell Phenotype Markers
The incompatibility of the TMB method with immunocytochemical determination of cell phenotype has limited the amount of information obtained from tract tracing studies using this histochemical procedure for HRP visualization. Therefore, the identification of trans-mitter phenotype of traced or innervated neurons, synaptic contact establishment by the traced cells, and/or neuronal activity, as indicated by functional markers, requires alternative approaches. Such methods either can include additional steps in tissue processing to stabilize the TMB reaction product (
In this report we have demonstrated that co-localization of the HRP tracer and cell phenotype markers can be achieved by double immunofluorescence. Although the immunofluorescence technique required a higher concentration of the anti-HRP antiserum because of its reduced sensitivity compared to the ABC detection systems, this method allowed the identification of transganglionically labeled motor neurons in the spinal cord with visualization of both perikarya and proximal neurites.
Taken together, these results provide further support for the immunocytochemical detection of HRP tracer as a sensitive alternative for the visualization of axonally transported HRP and the applicability of this method for the simultaneous identification of cell phenotype markers.
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Acknowledgments |
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Supported by National Institutes of Health Grant (NS33776), the Sid W. Richardson Foundation (GMS), and the Daniel Heumann Spinal Cord Foundation (MIR).
We are grateful to Jason Hale for helpful comments on this manuscript.
Received for publication October 8, 1998; accepted October 13, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:1457-1463
Ellis J, Halliday G (1992) A comparative study of avidin-biotin-peroxidase complexes for the immunohistochemical detection of antigens in neural tissue. Biotech Histochem 67:367-371[Medline]
Grumbach IM, Veh RW (1995) The SA/rABC technique: a new ABC procedure for detection of antigens at increased sensitivity. J Histochem Cytochem 43:31-37
Hsu SM, Raine L, Fanger H (1981) Use of avidinbiotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP). J Histochem Cytochem 30:577-580
Kressel M (1998) Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry. J Histochem Cytochem 46:527-533
LaMotte CC, Kapadia SE, Shapiro CM (1991) Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoid-HRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP). J Comp Neurol 311:546-562[Medline]
Lindh B, Aldskogius H, Hökfelt T (1989) Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig. Histochemistry 92:367-376[Medline]
Mesulam MM (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26:106-117[Abstract]
Mesulam MM, Hegarty E, Barbas H, Carson KA, Gower EC, Knapp AG, Moss MB, Mufson EJ (1980) Additional factors influencing sensitivity in the tetramethylbenzidine method for horseradish peroxidase neurohistochemistry. J Histochem Cytochem 28:1255-1259[Abstract]
Morrell JI, Greenberger LM, Pfaff DW (1981) Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics. J Histochem Cytochem 29:903-916[Abstract]
Myers JD (1988) Development and application of immunocytochemical staining techniques: a review. Diagn Cytopathol 5:318-330
Rye DB, Saper CB, Weiner BH (1984) Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry. J Histochem Cytochem 32:1145-1153[Abstract]
Schmidt ML, Trojanowski JQ (1985) Immunoblot analysis of horseradish peroxidase conjugates of wheat germ agglutinin before and after retrograde transport in the rat peripheral nervous system. J Neurosci 5:2779-2785[Abstract]
Vacca LL, Rosario SL, Zimmerman EA, Tomashefsky P, Ng P, Hsu KC (1975) Application of immunoperoxidase techniques to localize horseradish peroxidase-tracer in the central nervous system. J Histochem Cytochem 23:208-215[Abstract]
van der Want JJL, Klooster J, NunesCardozo B, de Weerd H, Liem RSB (1997) Tract-tracing in the nervous system of vertebrates using horseradish peroxidase and its conjugates: tracers, chromogens, and stabilization for light and electron microscopy. Brain Res Protocols 1:267-279
Waar WB, de Olmos JS, Heimer L (1981) Horseradish peroxidase: the basic procedure. In Heimer L, Robards MJ, eds. Neuroanatomical Tract Tracing Methods. New York, Plenum Press, 207-262
Wan XS, Trojanowski JQ, Gonatas JO (1982) Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: their uptake and clearance, transganglionic and retrograde transport, and sensitivity. Brain Res 243:215-224[Medline]
Watson RE, Jr, Wiegand SJ, Clough RW, Hoffman GE (1986) Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7:155-159[Medline]