ARTICLE |
Correspondence to: Stephen A. Schnell, 4-102 Owre Hall, Dept. Cell Biology and Neuroanatomy, 321 Church Street SE, U. of Minnesota, Minneapolis, MN 55455.
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Summary |
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The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 110 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels. (J Histochem Cytochem 47:719730, 1999)
Key Words: copper sulfate, Sudan Black B, monkey, human, primate, rat, immunohistochemistry, immunocytochemistry, retrograde axonal tract tracing
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Introduction |
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As an animal ages, the autofluorescent pigment lipofuscin accumulates in the cytoplasm of many cell types, including those of the CNS (
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Materials and Methods |
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Experimental Animals, Retrograde Tract Tracing, and Tissue Preparation
Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. Tissue from two male Rhesus monkeys (Macaca mulatta, 45 kg body weight; Sierra Biomedical, Sparks, NV) were used in these studies. To study the effect of lipofuscin reduction on retrograde tract tracers, several tracers were injected into either monkeys or rats. The monkeys were maintained under isofluorane anesthesia and an incision was made at the level of the lumbar enlargement. A vertebra was partially removed to expose the spinal cord. A syringe (Hamilton Gastight; Reno, NV) equipped with a glass micropipette filled with retrograde tracer was introduced into the dorsal spinal cord. One monkey was injected with a mixture of 2% True Blue and 2% Fast Blue (Sigma Chemical; St Louis, MO); the other was injected with a 2% wheat germ agglutininhorseradish peroxidase (WGA-HRP; Sigma) solution. Injection micropipettes were pulled from 1.5 mm outer diameter, 1.2 mm inner diameter glass capillary tubing using a micropipette puller (Narishige; Tokyo, Japan) and the tips trimmed to a diameter of ~50 µm. Pressure injections (100500 nl at a rate of ~100 nl/min) were made into each monkey; after each injection, the micropipette was held in place for 5 min to reduce backwelling through the pipette tract. The monkeys were allowed to survive 510 days before they were sacrificed by vascular perfusion after being deeply anesthetized (10 mg/kg ketamine, 0.1 mg/kg butorphanol, and 0.5 mg/kg acepromazine). The monkeys were first perfused with calcium-free Tyrode's solution (116 mM NaCl, 5 mM KCl, 2 mM MgCl2, 400 µM MgSO4, 1.2 mM NaH2PO4, 26 mM NaHCO3, and 2.9 mM glucose), followed by fixative (4% w/v depolymerized paraformalde-hyde in 160 mM phosphate buffer containing 14% v/v saturated aqueous picric acid, pH 6.9). After fixation, the animals were perfused with 5% w/v sucrose in 100 mM phosphate buffer, pH 7.2. The brains and spinal cords were removed and immersed in 5% sucrose overnight before sectioning. Tissue was sectioned either on a cryostat (Bright Instruments; Huntington, UK) at 10 µm and stored at -20C or on a freezing microtome (Leitz Instruments; Heidelberg, Germany) at 2050 µm and stored in PBS at 4C for 23 days.
Two SpragueDawley rats (Harlan; Madison, WI) were used in this study. One rat (150 g body weight) was anesthetized with 75 mg/kg ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine. Hypoglossal motoneurons in this rat were retrogradely labeled by injection into each side of the tongue with 500 nl 10% hydroxystilbamidine (
A waste portion of one human medulla was obtained under a protocol approved by the University of Minnesota Institutional Review Board. Sections were cut at a nominal thickness of 20 µm on a freezing microtome.
Immunocytochemistry
Sections were immunofluorescently labeled using either goat anti-calcitonin gene-related peptide diluted 1:200 (CGRP; a gift from Hunter Heath, Mayo Clinic, Rochester, MN), goat anti-serotonin diluted 1:100 (5-HT, 5-hydroxytryptamine, (
Attempted Reduction of Lipofuscin Autofluorescence
After review of the literature regarding the nature and composition of lipofuscin, several protocols were used in an attempt to reduce or eliminate the autofluorescence of lipofuscin. These included treating tissue sections with potassium permanganate alone (
Histochemical Protocols to Reduce Lipofuscin Autofluorescence
Cupric Sulfate.
After immunocytochemistry, the sections were removed from the PBS wash, dipped briefly in distilled H2O, and treated with CuSO4 (Fisher Scientific; Pittsburgh, PA) in ammonium acetate buffer (50 mM CH3COONH4, pH 5.0) for 1090 min, dipped briefly in distilled H2O, and returned to PBS. The sections were either mounted with PBS/glycerol containing 0.1% p-phenylenediamine (PPD; 5.0). Although that group included 100 µM ethylendiamine tetra-acetic acid in the ammonium acetate buffer, our preliminary experiments determined that this compound was unnecessary for successful reduction of lipofuscin autofluorescence.
Sudan Black. After immunocytochemistry, the sections were treated with a solution of Sudan Black B (Allied Chemical; New York, NY) in 70% methanol for 5 min. If overstained, they were then differentiated by dipping in clean 70% ethanol until a desired level of staining was achieved and then were mounted with PBS/glycerol/PPD. Sudan black histochemical staining was not compatible with xylene-based mounting media (e.g., DPX) because SB is lipophilic and is removed from tissue on immersion in xylene.
Microscope and Filter Systems
The sections were examined with an Olympus BH-2 microscope equipped for reflected fluorescence illumination and digital imaging. Filter bandpasses were as follows: FG or True BlueFast Blue (wideband UV) 330390-nm bandpass excitation filter and a 420-nm longpass emission filter; FITC/Cy2 (green fluorophores) 460490-nm excitation and 510550-nm emission; rhodamine/Cy3 (red fluorophores) 541551-nm excitation and 572607-nm emission; and Cy5 (deep red fluorophore) 615635-nm excitation and 655-nm longpass emission. Black-and-white digital images were collected with a Cohu 4915 CCD camera (Cohu; San Diego, CA) and Power Macintosh 7100 computer (Apple Computer; Cupertino, CA) equipped with an image acquisition board (model LG-3; Scion Image, Frederick, MD) and using Scion Image version of the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih/image/). Color digital images were obtained using a cooled color CCD camera (Optronix; Schaumberg, IL) and acquired with MetaMorph software (Universal Imaging; West Chester, PA). Digital images were manipulated with Photoshop 5.0 software (Adobe Systems, San Jose, CA) using a Power Macintosh G3 computer and were printed with a Pictrography 3000 color printer (Fujix; Tokyo, Japan).
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Results |
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General Observations
Lipofuscin-like autofluorescence was found throughout the neuraxis of macaques, including cortex, hippocampus, cerebellum, thalamus, hypothalamus, medulla, spinal cord, and dorsal root ganglia (Figure 1). There appeared to be lipofuscin-like pigments in cells of all sizes in most of these regions, although not all cells emitted autofluorescence. For example, in the cerebellum the majority of lipofuscin-like autofluorescence was observed surrounding Purkinje cells, whereas less was observed within the Purkinje cells themselves. Furthermore, little or no autofluorescence was observed in cells comprising the molecular and granular layers of the cerebellum.
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The excitation and emission characteristics of the lipofuscin-like autofluorescence were sufficiently broad that they could complicate the use of fluorescence techniques (Figure 2). Lipofuscin-like autofluorescent pigments were visible under fluorescent filters for UV, fluorescein, rhodamine, and Cy5. Using the UV filter, the lipofuscin-like fluorophore was punctate and gold-yellow, which is similar in appearance to FG. Using other filters, it appeared either green (using the filters for fluorescein), red (using the filters for rhodamine), or deep red (using the filters for Cy5); the intensity of the lipofuscin-like compound could be as strong as that of the strongest immunofluorescence. Thus, lipofuscin-like autofluorescence could mimic the appearance of immunofluorescent labeling (Figure 3). In addition to the histochemical means to reduce lipofuscin autofluorescence mentioned above (see Materials and Methods), we attempted to reduce it using high-intensity, long-duration UV illumination (x60, 1.4 NA objective, 1 hr duration. Mounting medium was omitted from under the coverslip to allow free access of molecular oxygen). The lipofuscin-like fluorophore did not photobleach to an appreciable extent (Figure 4).
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Effect of CuSO4 or Sudan Black Treatment on Lipofuscin-like Autofluorescence
Depending on the concentration used, CuSO4 treatment greatly reduced or eliminated the lipofuscin-like autofluorescence in monkey spinal cord (Figure 5). At low concentrations (1100 µM CuSO4), grains of yellow autofluorescent pigment could still be observed in neuronal cytoplasm. At higher concentrations (110 mM CuSO4), almost all of the autofluorescent material was eliminated from neuronal somata (Figure 5), although some yellow fluorescent material remained surrounding structures that resembled blood vessels (not shown). At the highest concentrations of CuSO4 (100 mM), all lipofuscin-like pigments were eliminated.
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CuSO4 is composed of two ions, either or both of which might be responsible for its action. To determine which ion (Cu2+ or SO42-) was the active component, we treated tissue with either cupric chloride or sodium sulfate in ammonium acetate buffer as above. Incubation of monkey spinal cord sections in 10 mM CuCl2 eliminated the lipofuscin-like autofluorescence (Figure 6). However, incubation of tissue sections in 10 mM Na2SO4 had no effect on lipofuscin-like autofluorescence (Figure 6).
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SB treatment of monkey CNS tissue also appeared to reduce or eliminate lipofuscin-like autofluorescence in a concentration-dependent manner (Figure 7). We found that concentrations of less than 1% SB were not sufficient to eliminate autofluorescent pigments. At the highest concentrations, 110% SB, all autofluorescent pigments were eliminated. To demonstrate the concentration-dependent effect of SB, the sections from the dilution series shown in Figure 7 were not destained (see Materials and Methods).
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On the basis of the above experiments with monkeys, we tested the efficacy of the CuSO4 and SB treatments using CNS tissue collected from one elderly human and one rat (aged 7 months). In human medulla, CuSO4 treatment provided substantial reduction of lipofuscin-like autofluorescence. However, autofluorescence in many large cells of the inferior olivary nucleus remained. SB treatment was able to eliminate lipofuscin-like fluorescence in human tissue (Figure 8). In the rat, both treatments successfully eliminated the autofluorescent pigments (Figure 8).
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Effect of CuSO4 or Sudan Black Treatment on Immunofluorescent Labeling
In preliminary experiments, we found that pretreatment of tissue by CuSO4 or SB before (rather than after) immunocytochemistry unacceptably reduced the intensity of immunocytochemical labeling of monkey tissue. Therefore, tissue treatment with SB or CuSO4 was performed after the final PBS wash (i.e., after the final rinsing of secondary antibodies). We found that CuSO4 reduced the intensity of immunofluorescence in a concentration-dependent manner. However, the effect of CuSO4 on lipofuscin was much more potent than its effect on immunofluorescence (Figure 9). At low concentrations (50 µM5 mM), the deleterious effect on immunofluorescence was much less pronounced; the intensity of immunocytochemical labeling was reduced but was readily visible. However, at the highest concentrations, (50 mM), it was difficult to visualize any of the immunocytochemical labeling.
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Sudan Black treatment also reduced immunofluorescence in a concentration-dependent manner (Figure 10). Concentrations of 1% SB allowed visualization of all the fluorophores tested, whereas only Cy3 could be visualized at the highest concentration (10% SB).
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Effect of CuSO4 or Sudan Black Treatment on Retrograde Tract Tracing
We also attempted to determine whether it was practical to reduce lipofuscin-like fluorophores using CuSO4 and SB in experiments in which fluorescent retrograde tract tracing dyes were used. In the rat, both CuSO4 and SB induced a slight reduction in the intensity of FG retrograde labeling in the hypoglossal nucleus (Figure 11). In monkeys, retrograde labeling of dorsal root ganglion cells by a mixture of True Blue and Fast Blue was slightly reduced using CuSO4 but completely eliminated using SB (Figure 11). Retrograde labeling of monkey bulbospinal neurons by WGA-HRP (observed using immunofluorescent labeling for WGA) was affected to the same degree as for immunocytochemical labeling (not shown, but see above).
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Discussion |
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The interpretation of fluorescence microscopy of tissue from older animals in general, and from monkeys and humans in particular, has been made difficult by the presence of the autofluorescent pigment lipofuscin, which accumulates in the cytoplasm of cells as animals age. We and others (
Because of the difficulties presented by lipofuscin-like autofluorescence, we were interested in finding a protocol that eliminated or reduced it and that was compatible with our fluorescent immunocytochemical and retrograde tract tracing procedures. Previously, it was suggested that lipofuscin-like autofluorescence could be reduced by the use of younger animals (
Previous studies have reported that lipofuscin could be extracted with a mixture of chloroform and methanol (
In the latter studies, it was found that treatment of the aqueous extract with certain metallic salts decreased or eliminated the yellow fluorescence, depending on the pH of the solution used (
In agreement with recent studies (
The little that is known regarding the chemical nature of lipofuscin has been reviewed by
In summary, the experiments described here demonstrated that two methods that reduced the intensity of lipofuscin-like fluorophores in monkeys were compatible with common fluorescent immunocytochemical and retrograde tract tracing techniques. Substantial reductions of lipofuscin-like autofluorescence in monkey CNS tissue sections were obtained with CuSO4 or Sudan Black. Furthermore, these substances were shown to be compatible with a wide range of fluorophores commonly used for immunocytochemical labeling. Although CuSO4 or SB also reduced the intensity of immunocytochemical labeling, this reduction did not appear to decrease our ability to visualize specific labeling. In fact, the reduction of immunocytochemical labeling could be largely overcome by using longer exposures while imaging. The two techniques had differential effects on the fluorescence of retrograde tract tracers. In the rat, CuSO4 or Sudan Black slightly attenuated the intensity of FG labeling. However in monkeys, a mixture of True Blue and Fast Blue used as a retrograde tract tracer showed that CuSO4 only reduced the intensity of the specific labeling but SB completely eliminated it.
We conclude that treatment of monkey tissue with CuSO4 or SB strikes an acceptable balance between reduction of lipofuscin-like autofluorescence and retention of immunocytochemical labeling. Because SB is incompatible with some fluorescent retrograde tract tracers and with xylene-based permanent mounting media, we also conclude that the cupric sulfate protocol may be of more general utility for the reduction of lipofuscin-like autofluorescence. However, SB may be superior for eliminating lipofuscin-like autofluorescence in aged human CNS tissue.
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Acknowledgments |
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Supported by PHS grant DA09642 from NIDA.
Received for publication February 17, 1999; accepted February 23, 1999.
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