ARTICLE |
Correspondence to: Victoria L. Singer, Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402.
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Summary |
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We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:14431455, 1999)
Key Words: ELF, phosphatase, fluorescence, endogenous activity, histochemistry
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Introduction |
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The highly differential yet almost ubiquitous nature of alkaline phosphatase (EC 3.1.3.1) expression has implicated it in a variety of embryological, developmental, and pathological processes (
Alkaline phosphatase histochemistry was first demonstrated with the calcium phosphate precipitation method of
The problems of reaction product diffusion and high background have persisted because most enzyme substrates yield soluble, colorless hydrolysis products that must be coupled with a salt or other capture reagent to generate a colored or fluorescent precipitate. Diffusion of the reaction product away from the site of enzymatic activity before it precipitates can compromise resolution. The presence of a salt or capture reagent in the reaction mixture can increase background fluorescence and lower specificity.
Another fluorescence-based method employed calcium binding fluorophores, such as calcein, in a Gomori-type technique (
We describe a fluorescence-based technique for detecting endogenous alkaline phosphatase activity that utilizes a unique fluorogenic substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, or ELF (Enzyme-Labeled Fluorescence)-97 phosphate. The ELF-97 alcohol liberated from the substrate forms a bright yellow-green fluorescent precipitate at the site of enzymatic activity, which is directly visible by fluorescence microscopy (
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Materials and Methods |
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Reagents
Dulbecco's modified Eagle's medium (DMEM), L-gluta-mine, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), gentamicin, and fetal bovine serum (lot #1004931) were from Life Technologies (Grand Island, NY). Dulbecco's PBS, Tween-20, Triton X-100, levamisol, L-phenylalanine, naphthol AS-MX phosphate, Fast Red TR salt, and Fast Blue BB salt were from Sigma (St Louis, MO). 5-Bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), and bovine serum albumin (BSA) were from Boehringer Mannheim (Indianapolis, IN). Hoechst 33342, ELF-97 Endogenous Phosphatase Detection Kit, ELF spin filters, Texas Red-X wheat germ agglutinin conjugate, and Alexa 594 goat anti-mouse IgG F(ab')2 fragment conjugate were from Molecular Probes (Eugene, OR).
Tissue
Wild-type adult zebrafish (Brachydanio rerio) were fixed for 6 hr at 4C in 3.7% paraformaldehyde, 100 mM phosphate, 0.15 mM CaCl2, 4% sucrose, pH 7.3. Afterwards, the tissue was washed in phosphate buffer and stored overnight at 4C in 30% sucrose. The tissue was embedded in Tissue-Tek OCT (Sakura Finetek USA; Torrance, CA) and frozen in liquid nitrogen. Sixteen-µm thick cryosections were placed on gelatin-coated slides.
Cells
Rat osteoblastic osteosarcoma cells UMR-106 (ATCC #CRL-1661; Rockville, MD) were grown on coverslips in DMEM with 2 mM L-glutamine, 10 mM HEPES, 0.5% (v/v) gentamicin, and 10% fetal bovine serum. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37C. After growth, the cells were rinsed three times in PBS (pH 7.4), then treated with 3.7% formaldehyde in PBS for 10 min, and then rinsed three times in PBS before storage at 4C.
Alkaline Phosphatase Histochemistry
In preparation for alkaline phosphatase histochemistry, tissue sections and fixed cultured cells were soaked in 0.2% Tween-20/PBS for 10 min and then rinsed in PBS for 10 min. Solubilization of membranes with nonionic detergent increases detectable alkaline phosphatase activity (
Histochemistry with the ELF-97 phosphate was performed with the ELF-97 Endogenous Phosphatase Detection Kit. The ELF-97 phosphatase substrate was diluted 1:201:40 in ELF-97 Developing Buffer (provided in the kit) and then filtered through an ELF spin filter. The reaction mixture was applied to prepared sections and signal development was monitored at the fluorescence microscope.
For azo dye histochemistry (
For histochemistry with BCIP/NBT (
The calcium phosphate precipitation method (
Staining with Antibody and Labeled Binding Protein
For staining with Texas Red-X wheat germ agglutinin conjugate, sections were first blocked (30 mM Tris-Cl, 150 mM NaCl, 1% BSA, 0.5% Triton X-100, pH 8.0) for 30 min and then washed (30 mM Tris-Cl, 150 mM NaCl, 1% BSA, 0.05% Triton X-100, pH 8.0) for 30 min. Texas Red-X wheat germ agglutinin conjugate (1 mg/ml) was diluted 1:150 in wash buffer, applied to the samples, and incubated for 10 min in a humid chamber. Slides were then washed in PBS for 10 min. Samples were soaked in 0.2% Tween-20 in PBS for 10 min and then rinsed in PBS for 10 min. Alkaline phosphatase histochemistry with the ELF-97 phosphate was then performed as above. In control staining experiments the labeled wheat germ agglutinin was omitted.
Immunohistochemistry with the RBM211.13 monoclonal antibody directed against alkaline phosphatase (the kind gift of Dr. Jane E. Aubin, University of Toronto) was performed on UMR-106 cells fixed as described above. Because treatment with Tween-20 did not allow antibody penetration, cells were permeabilized in 100% acetone (-20C) for 5 min and then rinsed in PBS. Cells were then blocked (1% BSA, 0.1% Tween-20, PBS) for 30 min. The antibody was diluted 1:250 in blocking buffer and applied to the cells. Samples were incubated for 30 min in a humid chamber. Cells were then washed five times, 5 min each, in PBS. The secondary antibody, Alexa 594 goat anti-mouse IgG F(ab')2 fragment conjugate, was diluted 1:200 in blocking buffer and incubated on the cells for 30 min in a humid chamber. Cells were then washed five times, 5 minutes each, in PBS. For double labeling experiments with RBM 211.13 and the ELF-97 phosphate, cells were equilibrated in ELF-97 developing buffer after the PBS washes; the reaction mixture containing the substrate was then applied as above. In control staining experiments the primary antibody was omitted.
Photostability
Data from the photobleaching studies were acquired through a x60/1.40 NA objective lens (Nikon; Melville, NY) with a cooled CCD camera (Quantix; Photometrics, Tucson, AZ) using the optical filter sets described below. Labeled tissues were mounted in PBS; no antifade agents were used. Briefly, while focused on a single tubule cross-section, the threshold was adjusted to the brightest field and the intensity measured to compute the average gray value. Samples were exposed to constant UV illumination for 90 sec, during which images were acquired at 5-sec intervals. Data were analyzed for fluorescence intensity as a function of time using an image processor (Metamorph; Universal Imaging, West Chester, PA). Three data sets for the ELF-97 alcohol precipitate and the Fast Red azo dye adduct, representing different fields of view, were averaged to obtain the plots.
Signal Visualization and Photography
All photography was performed with a Nikon Labophot fluorescence microscope using filter sets from Omega Optical (Brattleboro, VT). The ELF-97 signal was visualized with either an ELF-97 bandpass filter set (excitation 365 ± 8 nm; emission 515 nm) or a Hoechst/DAPI longpass filter set (excitation 365 ± 8 nm; emission
400 nm). Hoechst dye signal was viewed through an AMCA bandpass filter set (excitation 365 ± 8 nm; emission 450 ± 33 nm). Fast Red TR azo dye adduct, Texas Red conjugate, and Alexa 594 conjugate signals were visualized through a Texas Red bandpass filter set (excitation 560 ± 20 nm; emission 635 ± 28 nm). Double exposures of ELF-97 precipitate and Hoechst dye signals were performed with the ELF-97 and the AMCA bandpass filter sets. Double exposures of the red fluorescent signals, ELF-97 precipitate, and Hoechst dye were performed with the Texas Red bandpass and Hoechst/DAPI longpass filter sets. Ektachrome Elite 400 color slide film was used. Exposure time was typically 13 sec for ELF-97 precipitate signals, 35 sec for Hoechst dye, and 13 sec for the Fast Red TR azo dye adduct, Texas Red conjugate, and Alexa 594 conjugate signals. Equal exposure times were used for fluorescence images of negative controls.
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Results |
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We used the ELF-97 phosphatase substrate, the Gomori method, BCIP/NBT, and naphthol AS-MX phosphate with either Fast Red TR (fluorescent) or Fast Blue BB (colored) salt to detect endogenous alkaline phosphatase activity in cryosections of adult zebrafish. Each technique was optimized with respect to concentration of reaction components and duration of the labeling reaction to generate the best possible staining with the lowest possible background signal. Development of the signal was carefully monitored. The ELF-97 alcohol precipitate signal was optimal after 3090 sec and the reaction was stopped immediately. The Fast Red TR azo dye adduct stain was best after 510 min, whereas the Fast Blue BB azo dye adduct signal required 1015 min. Staining with BCIP/NBT gave optimal signal after 2030 min. The Gomori method gave the best labeling after a 3-hr incubation period with the substrate. We compared resolution, signal diffusion, and photostability of each of the methods.
In our preliminary studies, we examined the staining pattern obtained with the calcium phosphate precipitation technique (
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Resolution
To compare resolution, we examined the localization of endogenous alkaline phosphatase activity to the brush border of the intestinal epithelium. The polarized columnar enterocytes that constitute this simple epithelium are characterized by many microvilli on their apical surface, too small to be resolved by light microscopy and collectively referred to as the brush border (
Diffusion
To investigate signal diffusion, we examined detection of endogenous alkaline phosphatase activity in the epithelium covering the ovary. This thin epithelium provided a very narrow, tightly localized band of alkaline phosphatase activity that, when labeled, permitted observation of any diffusion of signal away from the sites of enzyme activity. All methods detected endogenous alkaline phosphatase activity in a thin layer cells on the surface of the ovary (Figure 3A3D). We found that the fluorescent ELF-97 precipitate was localized to the epithelium and showed no signs of diffusion; no signal was observed lateral to the epithelium, neither deep (towards the yolk platelets) nor away from the surface of the cells (Figure 3E). In contrast, samples stained by the other methods all showed signals lateral to the epithelial cells (Figure 3F3H). Compared to the narrow band of labeling produced by ELF-97 precipitate, staining generated by the other methods was wider and more diffuse (compare Figure 3E to Figure 3F3H). Little or no nonspecific labeling was observed for any of the methods. Weak autofluorescence caused by yolk platelets was observed (Figure 3I and Figure 3J). No labeling was detected in control samples incubated with the alkaline phosphatase inhibitor levamisol (Figure 3I3L) (
Photostability
To compare the relative photostability of the signals generated by the two fluorescent detection methods, the ELF-97 precipitate and the Fast Red TR azo dye adduct, we examined the staining of specific tubules in the kidney. The proximal convoluted tubules of the kidney express a high level of alkaline phosphatase activity (
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Specificity
To further study the specificity of the ELF-97 phosphatase substrate, we examined its ability to accurately distinguish between adjacent expressing and nonexpressing cells. Tissue sections stained for alkaline phosphatase activity with the ELF-97 phosphate were counterstained with the blue fluorescent Hoechst 33342 nucleic acid stain to allow identification of cells known to lack endogenous alkaline phosphatase expression. In the intestine, the ELF-97 precipitate was localized to the brush border of the enterocytes and was absent from the invaginated region of the mucosa at the bases of the villi (Figure 6A, area between the arrows). This observation is consistent with findings that the invaginated mucosa, or intestinal crypt, contains undifferentiated enterocytes that do not express alkaline phosphatase (
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Compatibility
We next asked if staining for endogenous alkaline phosphatase activity with the ELF-97 phosphate was compatible with multiparameter assays employing labeled antibodies or binding proteins. The application of such reagents requires that nonspecific binding sites within the tissue be coated or blocked by incubation in a solution of BSA or serum. However, saturating the entire tissue with excess protein alters the immediate surroundings of endogenous enzymes and, consequently, might affect the detection of phosphatase activity by influencing the precipitation of the hydrolysis product. The poor crystallization of some azo dye adducts has been attributed to the environment of the enzyme (
Co-localization
We next examined the use of the ELF-97 phosphate for detecting endogenous alkaline phosphatase activity in cultured cells. Cultured UMR-106 osteosarcoma cells were permeabilized with 0.2% Tween-20, stained for endogenous alkaline phosphatase activity with the ELF-97 phosphate, and then counterstained with the Hoechst nucleic acid stain. We found that the ELF-97 signal was localized not only to the cell surface but also to a perinuclear location within the cell (Figure 8A, arrow). This finding, which was also observed in ROS 17.1 osteogenic cells (not shown), was unexpected because alkaline phosphatase is generally believed to be located on the cell surface (
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Discussion |
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Comments on the Endogenous Phosphatase Activity Detection Data
In two osteogenic cell lines, UMR-106 and ROS 17.1, we found that the ELF-97 precipitate localizes endogenous alkaline phosphatase activity to the plasma membrane as well as to a perinuclear site. Alkaline phosphatase is generally located on the cell surface, linked to the cell membrane via a phosphatidylinositol glycan linkage (
In cultured UMR-106 cells, labeling with ELF-97 phosphate did not produce a staining pattern identical to that produced with antibody labeling. However, there are areas in which the two signals co-localize. Most notable is the granular or punctate appearance of the ELF-97 precipitate on the cell surface, whereas antibody labeling is smoother and more continuous in the same region. We know that the punctate nature of this signal is not due to overstaining (see below) because the labeling pattern occurs early in the labeling time course. It is possible that the punctate staining pattern is due to localized differences in enzyme activity level, or that there are localized nucleation sites for precipitate formation in the membrane surface, perhaps due to its physical properties. The enzyme might be present uniformly in the cell membrane but is only active or is most active in specific localized regions. We observed that in cells that were first labeled with the antibody and then assayed for enzymatic activity, signals took more time to fully develop and were generally weaker than signals obtained from cells that were not treated with the antibody. These observations confirm that the antibody is able to bind the same enzyme as that required for substrate turnover.
General Observations on Achieving Optimal Results with ELF-97 Phosphate in This Application
Our studies show that use of the ELF-97 alkaline phosphatase substrate to detect endogenous phosphatase activity in tissues and cells can produce a well-localized fluorescent signal that resists diffusion and exhibits little or no background fluorescence. However, as is the case for most enzyme-mediated histochemical reactions, obtaining optimal results with this reagent requires careful monitoring of the signal development process. Excessive incubation times, which result in overstaining, can result in lower than optimal target resolution and increased nonspecific background staining. Because the ELF-97 precipitate is very photostable (
One other common cause of spurious background crystals on the slides is omission of the substrate filtration step recommended by the manufacturer. This step removes spontaneous hydrolysis products of the substrate that are sometimes present in the stock solution. These hydrolysis products are sometimes themselves large enough crystals to give rise to background signals and at other times appear to act as nucleation sites for spurious crystal formation on the sample. Use of the recommended mounting medium also appears critical, both for reducing background signals and for maintaining optimal signal levels. Enzymatic hydrolysis of the ELF-97 phosphate yields an alcohol precipitate that is not stable in glycerol-based mounting media. Partial dissolution of this precipitate into glycerol-based media causes an immediate signal decrease and also can lead to the deposit of spurious crystals from the site of enzymatic activity to other regions of the sample.
We have also found that signal granularity is in some cases related to the surface properties of the sample. In zebrafish brain cryosections, for example, we observed that the substrate sometimes gives rise to extremely granular or punctate signals on some surfaces, whereas others label in such a way that it appears that the precipitate is smoothly painting the cell surfaces. We believe that the specific molecular composition of certain tissues, including the carbohydrate or lipid content of those tissues, might be the cause of this sort of problem.
Finally, although several researchers have published on the use of standard fluorescein photographic filters and excitation sources with the ELF-97 phosphatase substrate (
Comments on Optimizing Fast Red TR Azo Dye Techniques
In contrast to the observations of previous researchers (
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Conclusions |
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In conclusion, we have presented a fluorescence-based method for alkaline phosphatase histochemistry that offers several advantages over conventional colorimetric and fluorescence-based techniques. The technique is compatible with multiparameter assays utilizing antibodies and labeled binding proteins. We found that it is useful with many different tissues, including autofluorescent samples, and that it is compatible with a variety of embedding methods. The substrate has also recently been found to be useful in flow cytometry for detecting endogenous alkaline phosphatase activity in marine phytoplankton (
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Acknowledgments |
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We wish to thank Dr Jane E. Aubin (University of Toronto) for the RBM 211.13 antibody and helpful discussions. We also thank Karen D. Larison for developing the ELF-97 Endogenous Phosphatase Detection kit and performing the Gomori method work, Diane Gray for help with the photostability studies and cell cultures, Collette Gilliland for assistance with graphics, Violette Paragas for her pioneering work with the ELF-97 phosphate, and several Molecular Probes employees for critical review of the manuscript.
Received for publication December 16, 1998; accepted June 15, 1999.
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