ARTICLE |
Correspondence to: Maryann E. Martone, Dept. of Neuroscience, University of California, San Diego, La Jolla, CA 92093-0608.
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Summary |
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We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution. (J Histochem Cytochem 49:13511361, 2001)
Key Words: cytoskeleton, dendritic spines, electron tomography, 3D reconstruction, Purkinje cells
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Introduction |
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SEVERAL METHODS have been introduced to study the organization of actin networks at the light and electron microscopic levels, including structural methods (
Phalloidin has been primarily used in studies of F-actin in cultured cells using light microscopy. We have adapted the phalloidin staining technique for studies of F-actin in slices of brain tissue at the light and electron microscopic level through the use of the fluorescent tag eosin and fluorescence photo-oxidation. Although fluorescent molecules are not in themselves visible under the electron microscope, they can be used to drive the oxidation of diaminobenzidine (DAB) to create a reaction product that can be rendered electron dense (
We have used eosinphalloidin/photo-oxidation to study the distribution of actin-rich structures in the rat central nervous system at the electron microscopic level (
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Materials and Methods |
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Materials
Eosinphalloidin and rhodaminephalloidin, phalloidinAlexa- 488, and Alexa568 were obtained from Molecular Probes (Eugene, OR). 3,3'-Diaminobenzidine tetrahydrochloride (DAB), coldwater fish gelatin, and the anti-ß actin antibody were purchased from Sigma (St Louis, MO). Paraformaldehyde, EM grade glutaraldehyde, sodium cacodylate, and Durcupan ACM resin were obtained from Electron Microscopy Sciences (Ft Washington, PA). Special glass bottom-welled tissue culture plates were obtained from MatTek (Ashland, MA). Cell-Tak adhesive was obtained from Collaborative Research (Bedford, MA).
Tissue
We used seven adult male SpragueDawley rats in this study. Briefly, intracardiac perfusion was performed under deep anesthesia (containing 50 mg/kg ketamine, 1 mg/kg rhompun, and 5 mg/kg acetopromazine in sterile saline) with normal rat Ringer's at 35C, followed by fixative. For light microscopic analyses, rats were perfused with 4% formaldehyde (made fresh from paraformaldehyde) in cacodylate buffer, pH 7.2. For ß-antibody studies the animals were perfused with 4% paraformaldehyde, 0.1% glutaraldehyde. The brains were removed and fixed for 2 additional hours in the same solution at 4C. For electron microscopic studies, a range of fixative strengths was evaluated, containing either 2% or 4% formaldehyde or 0.5%2.5% glutaraldehyde. The tissue was postfixed for 2 hr in the same fixative. After removal of the brain from the skull, coronal or sagittal sections through neostriatum, cerebellum, and hippocampus were cut at a thickness of 5080 µm on a Leica Vibratome, model VT 1000E.
Cultured Cells
As a control, we also labeled cultured bovine aortic endothelial cells (BAECs) fixed using the same conditions as above. These cells possess characteristic bundles of actin filaments called stress fibers. Details about culturing methods are given in
Staining Sections with Phalloidin
Vibratome sections were washed with 50 mM glycinePBS containing 0.5% coldwater fish gelatin to block nonspecific binding. After 30 min of washing, sections were incubated with agitation in a solution of 4 U eosinphalloidin and 0.5% coldwater fish gelatin50 mM glycinePBS for 2 hr. For light microscopic studies, phalloidin conjugated to rhodamine was also used because of its superior fluorescent quantum yield. As a negative control, the eosinphalloidin was omitted.
Immunolabeling
After repeated rinsing in 0.1 M PBS, sections were blocked for 30 min in PBS containing 1% normal donkey serum, 1% bovine serum albumin, 1% coldwater fish gelatin, and 0.2% Triton X-100. Sections were placed in the primary antibody against ß-actin (dilution 1:100) in 0.1 x strength blocking buffer (working buffer) and incubated on a rotator overnight at 4C. After several washes in working buffer, a secondary antibody conjugated to FITC was applied for 1 hr at room temperature. For electron microscopic studies, an immunoperoxidase procedure was employed. After incubation in the primary antibody, the tissue was washed in PBS and incubated for 1 hr in biotinylated anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA). The tissue was washed and then incubated for 1 hr in the avidinbiotinhorseradish peroxidase complex (Vector) according to the manufacturer's instructions. The antigen was visualized by reaction with DAB, postfixed with 1% osmium tetroxide for 20 min, dehydrated in ascending alcohols, and flat-embedded in Durcupan (Fluka; Milwaukee, WI). Ultrathin sections were observed on a JEOL 100CX electron microscope.
Procedures for immunolabeling cultured cells for tubulin using secondary antibodies conjugated to eosin, followed by photo-oxidation, can be found in
Confocal Microscopy
Fluorescent microscopy was performed on a Zeiss Axiovert 35 M inverted light microscope using either a x63 1.4 NA or x40 1.3 NA objective lens. Fluorescent and transmitted light images were recorded using a laser scanning confocal attachment (MRC-1024; Bio-Rad Laboratories, Cambridge MA) and a kryptonargon laser.
Photo-oxidation
The procedure for photoconversion described here follows that of
Tissue sections labeled previously with eosinphalloidin were washed several times with cacodylate buffer and then mounted on glass-welled tissue culture dishes (see above) pretreated with Cell Tak adhesive. Slices were fixed again for 25 min with 2% glutaraldehyde in 0.1 M cacodylate buffer, rinsed in buffer for several minutes, and placed in 50 mM glycine. The appropriate areas were located with transmitted light and the pattern of fluorescent labeling was recorded using the confocal attachment at a low laser power setting. The samples were immersed for 10 min in the DAB solution at 4C bubbled with pure O2 and then irradiated under conventional epifluorescence using a 75-W Hg lamp and a fluorescein filter set. The DAB solution was changed every few minutes while the reaction proceeded. During this process, the fluorescence faded quickly and brownish reaction product began to appear in place of fluorescence. The reaction was followed using transmitted light. When brown reaction product first began to appear (usually between 68 min after beginning irradiation), the reaction was stopped by blocking fluorescence excitation.
Preparation of Samples for Electron Microscopy
After photo-oxidation, the samples were rinsed in 0.1 M sodium cacodylate buffer several times and incubated in 1% osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4, for 30 min. Some sections were fixed for 1 hr in 2.25% glutaraldehyde in cacodylate buffer with 0.2% tannic acid added. Osmication was done with 0.75% OsO4 in cacodylate buffer, pH 6, for 1 hr on ice according to the procedure described by
Electron Microscopy
Thin sections (80100 nm) and thick sections (0.51 µm) were cut with a Reichert Ultracut E ultramicrotome using glass knives. Thin sections were examined using a JEOL 100CX electron microscope at 80100 keV and thick sections were observed using a JEOL JEM-4000EX intermediate voltage microscope (IVEM) at 400 keV. One set of thin sections was post-stained with a combination of uranyl acetate and lead citrate. Methods for 3D reconstruction using serial sections and electron tomography can be found in
Injection of Fluorescent Phalloidin into Single Cells
Three male SpragueDawley rats were anesthetized and perfused transcardially with Ringer's solution followed by 4% paraformaldehyde in PBS. The brain was postfixed in toto at 4C for 3 hr in the same fixative. The cerebellum was cut into 100-µm-thick sagittal vibratome slices. The slices were stored in ice-cold PBS until they were used. The slices were viewed in cold PBS with an Olympus BX50WI infrared differential interference contrast/epifluorescent microscope (Olympus; Melville, NY) using a x60 water immersion objective. Sharp glass micropipettes were pulled on a vertical pipette puller (David Kopf Instruments; Tujunga, CA) using Omegadot capillary tubes (OD 1.00 mm, ID 0.58 mm; resistances ranged between 100 and 400 M). Purkinje cells were selected on the basis of appearance and filled with a solution containing both 10 mM phalloidinAlexa488 and Alexa568 in 200 mM potassium chloride using a glass micropipette controlled by Narishige micromanipulators by applying a 0.5-sec negative current pulse (1 Hz). Once the Purkinje cells were filled, as determined by epifluorescent illumination, the tissue slices were fixed in 4% paraformaldehyde for 1015 min. The slices were then mounted on glass slides and coverslipped in Gelvatol. Dual channel z-series were obtained of Purkinje cells containing phalloidinAlexa using a Biorad laser scanning confocal microscope (x63 Zeiss oil immersion, 1.4 N, 0.319 µm pixel size, 0.18-µm z-step). The confocal data sets were processed, merged, and analyzed using the Imaris visualization package (Bitplane; Zurich, Switzerland).
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Results and Discussion |
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Distribution of F-Actin in the Rat CNS
Because eosin possesses only 20% the fluorescence quantum yield of more conventional fluorophores, such as fluorescein and rhodamine, we first compared the overall fluorescence pattern obtained with phalloidin conjugated to eosin, rhodamine, and Alexa488 (Fig 1). Although the overall fluorescence was weaker, the patterns of staining obtained with the eosin conjugates were similar to those of the other fluorophores (Fig 1C vs Fig 1D).
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In tissue fixed with 4% paraformaldehyde, we observed the strongest staining in brain areas known to be rich in dendritic spines (Fig 1). In these brain regions, phalloidin labeling appeared as a dense background of bright punctate staining. As we reported in our previous study (
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Although staining was most concentrated and consistent in dendritic spines, we also observed intense and consistent staining in a subset of additional structures in the CNS, including the cerebellar glomeruli, certain axons and dendrites, and in some astrocytic processes (Fig 1 and Fig 3). Many of these structures also were stained with an antibody against ß-actin (Fig 3), although the pattern of staining was not identical between the two methods. For example, in the cerebellum labeling of glial cells was much more prominent in the immunolabeled material, whereas perivascular labeling was more notable in the phalloidin tissue (Fig 3). Differences between phalloidin and antibody labeling should be expected because phalloidin is specific for F-actin, whereas antibodies usually do not distinguish between F- and G-actin (-actin, which are differentially distributed in some structures (
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Fixation
Using the pattern of staining obtained with 4% paraformaldehyde as a baseline, we then tested a range of fixative strengths to determine the effect of fixation on the pattern of staining and the penetration of phalloidin into the tissue. Concentrations of glutaraldehyde up to 1% in 4% paraformaldehyde did not alter the overall pattern of staining and did not interfere with the penetration or fluorescence signal of phalloidin into the tissue, as evaluated by optical sectioning using the confocal microscope. A vertical section through the molecular layer of a 70-µm-thick hippocampal slice stained with eosinphalloidin is shown in Fig 4. Note the uniform punctate staining observed through the thickness of the section, even without the addition of detergents in the incubation solutions. Tissue fixed with higher concentrations of glutaraldehyde required twice as long to photo-oxidize, leading to a decrease in structural preservation.
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Photo-oxidation of EosinPhalloidin
Regions exhibiting a moderate to intense fluorescent signal with phalloidineosin readily photoconverted in the presence of DAB. As a test system for the sensitivity and resolution of the technique, we labeled cultured BAECs with phalloidineosin. As shown in Fig 4, the stress fibers are clearly labeled, as is the subcortical actin at the LM level. Fig 4C shows an electron micrograph taken with intermediate voltage electron microscopy of a 0.5-µm-thick section through the same cell pictured in Fig 4B after photo-oxidation. The photo-oxidized image shows a faithful correspondence (arrows in Fig 4B and Fig 4C) to the LM image with additional detail visible because of the higher resolution. A thin section through a heavily labeled actin bundle (arrow) is shown in Fig 4D. As we have reported previously (
In sections of brain tissue, we also found a good correspondence between the light and electron microscopic images. As described above, in a given brain region only a subset of structures showed appreciable labeling with phalloidin. For example, in the cerebellar cortex, the most intense labeling was observed in dendritic spines in the molecular layer and in the mossy fiber glomeruli and pinceau region in the granule cell layer and surrounding the vasculature (Fig 1A). Astrocytic processes surrounding the Purkinje cell soma were detectable at the LM level but were more weakly stained than these other structures (AST in Fig 1A), whereas the main dendrites of Purkinje cells and the cell somas of interneurons in the molecular layer showed only diffuse light labeling. The intensity of labeling at the LM level affected the consistency and intensity of photo-oxidation reaction product at the EM level. The most intensely stained structures, e.g., dendritic spines and cerebellar glomeruli, photoconverted readily and were observed in every section examined (Fig 5A and Fig 5B). Structures displaying only light diffuse labeling, such as major dendrites, did not show detectable labeling under the electron microscope (Fig 3D and Fig 5A).
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The overall distribution of F-actin agreed well with reports from structural and immunocytochemical studies (
The use of phalloidineosin plus photo-oxidation is very helpful in EM identification structures rich in actin filaments because LM examination can be used to identify such structures before sectioning for EM. Using purely structural methods to identify relatively sparse F-actin-rich structures at the EM level would be very difficult in a complex tissue such as brain. However, although we could determine which structures were rich in F-actin by the presence of photo-oxidized DAB, we could not resolve individual actin filaments in labeled structures in brain tissue. Actin filaments are sensitive to osmium fixation, which can destabilize actin filaments even after glutaraldehyde treatment (
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Reconstructions at the EM Level
At the LM level we observed penetration of phalloidineosin through the entire depth of the tissue without detergent pretreatment. However, photo-oxidation appeared to be restricted to the surface of the section and did not extend beyond 810 µm or so into the tissue. Photo-oxidation is dependent on the generation of reactive oxygen species (
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Three-dimensional reconstructions were also performed at higher resolution using electron tomography. We employed tomography to study the organization of actin networks in material prepared with tannic acid (see above) and photo-oxidation. Electron tomography has the advantage of producing computed slices through the volume which are thinner than can be prepared using physical sectioning (see discussion in
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Conclusions |
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Phalloidineosin is a useful method for studying structures rich in F-actin at both the light and electron microscopic levels. This technique has several advantages over antibody- and structure-based methods including (a) the ability to perform correlative light and electron microscopy studies, (b) excellent ultrastructural preservation due to the use of high concentrations of glutaraldehyde and the lack of detergent pretreatments, (c) the good spatial resolution of EM labeling compared to peroxide-based methods, and (d) its compatibility with 3D electron microscopic techniques. Phalloidineosin photo-oxidation thus offers a relatively simple technique for gaining additional information about actin-rich structures at high resolution using electron microscopic analysis.
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Acknowledgments |
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Supported by NIH grants RR04050, DC03192, and DA02854.
Received for publication May 1, 2001; accepted July 26, 2001.
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