Journal of Histochemistry and Cytochemistry, Vol. 47, 1443-1456, November 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

A High-resolution, Fluorescence-based Method for Localization of Endogenous Alkaline Phosphatase Activity

William G. Coxa and Victoria L. Singera
a Molecular Probes, Inc., Eugene, Oregon

Correspondence to: Victoria L. Singer, Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Conclusions
Literature Cited

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 47:1443–1455, 1999)

Key Words: ELF, phosphatase, fluorescence, endogenous activity, histochemistry


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Conclusions
Literature Cited

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 (McComb et al. 1979 ; Harris 1990 ). Alkaline phosphatase expression is a marker for primordial germ cells (reviewed in McLaren 1992 ; Lawson and Hage 1994 ), osteoblast differentiation and bone metabolism (reviewed in Aubin et al. 1995 ; Christenson 1997 ), phosphate starvation in bacteria (reviewed in Van Dien and Keasling 1998 ), yeast (reviewed in Oshima 1997 ), and marine phytoplankton (Gonzalez-Gil et al. 1998 ), and is coincident with several forms of malignancy (reviewed in Millan and Fishman 1995 ). Although alkaline phosphatases are generally classified by their shared ability to hydrolyze orthophosphate monoesters at alkaline pH, their physiological roles remain poorly understood. Nevertheless, the localization of alkaline phosphatase activity in normal and neoplastic tissues and cells continues to be an extremely important endeavor.

Alkaline phosphatase histochemistry was first demonstrated with the calcium phosphate precipitation method of Gomori 1939 . A significant advance was made thereafter with the development of the azo dye technique (Menten et al. 1944 ). Burstone 1960 , Burstone 1962 introduced noncoupling and coupling azo dye techniques in which the enzymatic reaction product is either directly detectable, without a coupling reagent, or can be visualized by coupling with a diazonium salt. With the finding that many of the substituted naphthol AS phosphates yielded fluorescent products, this work also introduced a new approach to enzyme histochemistry, i.e., visualization of enzyme localization by fluorescence, which permitted detection of very low levels of activity. Nevertheless, these studies suffered because the reaction product was not adequately localized due to diffusion. In more recent studies, the use of naphthol AS-BI or AS-MX phosphate and the Fast Red TR salt was shown to produce a relatively well-localized red fluorescent final reaction product (Dolbeare et al. 1980 ; Ziomek et al. 1990 ; Narisawa et al. 1992 ), but the high nonspecific background staining was a problem (Dolbeare et al. 1980 ; Raap 1986 ; Kamalia et al. 1992 ). The Fast Red Violet salt with naphthol AS phosphate was shown to exhibit little or no nonspecific staining (Kamalia et al. 1992 ) but also generated a granular signal that precluded high-resolution localization (Ziomek et al. 1990 ).

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 (Raap 1986 ; Murray and Ewen 1992 ). Calcein, however, exhibits the same drawbacks as its parent molecule, fluorescein, in that it photobleaches rapidly under intense illumination and its excitation and fluorescence emission maxima are not well separated (i.e., it has a small Stokes shift), limiting its usefulness in autofluorescent tissues. Therefore, an ideal fluorescence-based histochemical technique would provide both a specific, well-localized precipitate and a very photostable signal with a large Stokes shift.

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 (Singer et al. 1994 ). Previous work has employed the ELF-97 phosphate in enzyme-mediated labeling techniques including immunohistochemical (Larison et al. 1995 ), cytochemical (Brumbuck and Wade 1996; Paragas et al. unpublished observations), and mRNA in situ hybridization (Bueno et al. 1996 ; Currie and Ingham 1996 ; Jowett and Yan 1996 ; Paragas et al. 1997 ) applications. The ELF-97 precipitate was found to be more photostable than fluorescein and distinguishable from other fluorescent labels, sample autofluorescence, and pigmentation. We demonstrate here the use of the ELF-97 phosphate in a fluorescence-based technique for localizing endogenous alkaline phosphatase activity in tissues and cultured cells.


  Materials and Methods
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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 (Ey and Ferber 1977 ). Specific labeling protocols are described below. In general, the labeling reaction was stopped with a PBS solution (pH 8.0) containing 25 mM EDTA and either 5 mM levamisol or 30 mM L-phenylalanine. Control reactions contained either 5 mM levamisol or 30 mM L-phenylalanine. Omission of the substrate, the capture reagent, or both from the labeling reaction produced no labeling in all cases. Counterstaining was performed in stop buffer containing 0.125 µM Hoechst 33342. ELF-97 precipitate-labeled samples were mounted in ELF-97 Mounting Medium (provided in the kit). All other samples were mounted in anhydrous glycerol. No antifade mounting agents were used.

Histochemistry with the ELF-97 phosphate was performed with the ELF-97 Endogenous Phosphatase Detection Kit. The ELF-97 phosphatase substrate was diluted 1:20–1:40 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 (Bancroft and Hand 1987 ), the labeling reaction solution contained 5 mg naphthol AS-MX phosphate dissolved in 0.5 ml N,N-dimethylformamide and 10 mg Fast Red TR salt or Fast Blue BB salt. The solution was mixed and then brought to a final volume of 10 ml with 0.1 M Tris-Cl buffer (pH 9.2). The solution was mixed again, filtered through a Whatman No. 2 filter, and used immediately. Labeling reactions were performed in the dark. Formation of the Fast Blue BB azo dye adduct was viewed briefly at regular intervals by light microscopy. The Fast Red TR azo dye adduct was monitored by fluorescence microscopy. To minimize photobleaching, signal development was viewed briefly at regular intervals.

For histochemistry with BCIP/NBT (Sambrook et al. 1989 ), the labeling reaction solution contained 0.17 mg/ml BCIP and 0.33 mg/ml NBT in reaction buffer (100 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl2, pH 9.5). Labeling reactions were performed in the dark. Signal development was viewed briefly at regular intervals by light microscopy.

The calcium phosphate precipitation method (Gomori 1939 ) was performed as described (Pearse 1968 ), with a Tris buffer substituted for the diethylbarbiturate buffer.

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 1–3 sec for ELF-97 precipitate signals, 3–5 sec for Hoechst dye, and 1–3 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.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Conclusions
Literature Cited

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 30–90 sec and the reaction was stopped immediately. The Fast Red TR azo dye adduct stain was best after 5–10 min, whereas the Fast Blue BB azo dye adduct signal required 10–15 min. Staining with BCIP/NBT gave optimal signal after 20–30 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 (Gomori 1939 ). Several tissues, including ovary and kidney, were labeled with the black precipitate characteristic of this detection method, indicating the presence of endogenous alkaline phosphatase activity (Figure 1A and Figure 1B). The intestine was also labeled, but very poorly (not shown). Staining with the ELF-97 phosphatase substrate also localized endogenous alkaline phosphatase activity to these tissues (see Figure 3A and Figure 4A). In comparison, staining with the ELF-97 phosphate gave a similar overall pattern; however, localization by the ELF-97 alcohol precipitate was more specific and gave better resolution of labeled cells than did the Gomori technique. The Gomori method produced much more nonspecific staining and the signals were not well localized to individual cells. Staining with the ELF-97 phosphate gave no background fluorescent labeling and the signals were restricted to the epithelium covering the ovary and the proximal convoluted tubules of the kidney, which are known to express high levels of endogenous alkaline phosphatase (McComb et al. 1979 ; Harris 1990 ). Preliminary studies with the azo dye and BCIP/NBT techniques also yielded more specific staining of these tissues than did the Gomori method. For this reason, the azo dye and BCIP/NBT techniques were further compared to the ELF-97 phosphate method.



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Figure 1. Localization of endogenous alkaline phosphatase activity using the calcium phosphate precipitation technique of Gomori 1939 . Zebrafish cryosections of ovary (A) and kidney (B) stained using the Gomori method. Bar = 100 µm.



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Figure 2. Localization of endogenous alkaline phosphatase activity in the intestine by the ELF-97 alcohol precipitate, Fast Red TR azo dye adduct, Fast Blue BB azo dye adduct, and BCIP/NBT. Cryosections of the zebrafish intestine stained by the ELF-97 alcohol precipitate (A,E), Fast Red TR azo dye adduct (B,F), Fast Blue BB azo dye adduct (C,G), and BCIP/NBT (D,H). The ELF-97 alcohol precipitate was more concentrated on the luminal surface of the epithelium (E, arrowhead). Arrows (A–D) note the interspersed, oval-shaped goblet cells, which lack endogenous alkaline phosphatase activity. Controls (I–L) performed with 30 mM L-phenylalanine. M and N are light microscopic images of the controls, I and J, respectively. Bar: AD = 50 µm; EH = 25 µm; IN = 100 µm.



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Figure 3. Localization of endogenous alkaline phosphatase activity in the ovary by the ELF-97 alcohol precipitate, Fast Red TR azo dye adduct, Fast Blue BB azo dye adduct, and BCIP/NBT. Cryosections of the zebrafish ovary stained by the ELF-97 alcohol precipitate (A,E), Fast Red TR azo dye adduct (B,F), Fast Blue BB azo dye adduct (C,G), and BCIP/NBT (D,H). Autofluorescence due to yolk platelets can be seen in I and J. Controls (I–L) performed with 5 mM levamisol. M and N are light microscopic images of the controls, I and J, respectively. Bar: AD = 100 µm; EH = 25 µm; IN = 100 µm.



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Figure 4. Localization of alkaline phosphatase activity in the kidney by the ELF-97 alcohol precipitate and Fast Red TR azo dye adduct. Cryosections of the zebrafish kidney stained by the ELF-97 alcohol precipitate (A,C) and the Fast Red TR azo dye adduct (B,D). The ELF-97 alcohol precipitate was more concentrated in the lumen of the tubules (A,C, arrows). Controls (E,F) performed with 5 mM levamisol. G and H are light microscopic images of the controls, E and F, respectively. Bar: A,B = 50 µm; C,D = 25 µm; EH = 100 µm.

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 (McComb et al. 1979 ; Harris 1990 ). Alkaline phosphatase is expressed in the apical region of the enterocyte and is concentrated in the brush border (McComb et al. 1979 ; Ziomek et al. 1980 ; Harris 1990 ). Staining was performed with the ELF-97 phosphate, naphthol AS-MX phosphate with either Fast Red TR salt or with Fast Blue BB salt, and BCIP/NBT. All methods localized endogenous alkaline phosphatase activity to the intestinal epithelium in the apical region of the enterocytes as expected (Figure 2A–2D). Staining with the ELF-97 phosphate produced, in addition to the apical labeling, a brighter, more concentrated signal on the luminal surface of the enterocytes, characteristic of the high amount of alkaline phosphatase expressed in the brush border (Figure 2E, arrowhead). In contrast, staining by the other methods produced an evenly distributed labeling in the apical region of the cell that did not show a stronger signal in the brush border (Figure 2F–2H). Samples assayed with the ELF-97 phosphate had no detectable nonspecific fluorescent labeling. Samples stained using the other techniques also exhibited no visible background. None of the techniques yielded signal in the interspersed goblet cells (Figure 2A–2D, arrows), which lack alkaline phosphatase expression (McComb et al. 1979 ; Wheater et al. 1987 ; Harris 1990 ). Furthermore, none of the techniques produced staining in controls incubated with the intestinal-type specific alkaline phosphatase inhibitor L-phenylalanine (Figure 2I–2L) (Goldstein et al. 1980 ).

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 3A–3D). 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 3F–3H). 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 3F–3H). 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 3I–3L) (Goldstein et al. 1980 ).

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 (McComb et al. 1979 ; Harris 1990 ) and, when labeled with fluorescence-based techniques, provided not only a very focused and intense signal but also a consistent cross-sectional area, which was ideal for measuring photostability. Tissue sections were stained for endogenous alkaline phosphatase activity with either the ELF-97 phosphate or the Fast Red TR azo dye technique (Figure 4A–4D). Staining with the ELF-97 phosphate produced labeling that was not only restricted to the proximal convoluted tubules but was more concentrated in the lumen, characteristic of the high amount of alkaline phosphatase expressed in the brush border of these tubules (Figure 4A and Figure 4C, arrows). Staining with the Fast Red TR azo dye adduct also produced signals exclusive to the proximal convoluted tubules but, in contrast to the ELF-97 signal, the labeling was fairly evenly distributed (Figure 4B and Figure 4D). Little or no nonspecific labeling was observed for either method. No labeling was observed in controls incubated with levamisol (Figure 4E–4H). To assay photostability, we illuminated cross-sections of labeled kidney tubules for 90 sec with constant, high-power UV light, acquiring images every 5 sec with a cooled CCD camera. The normalized data (Figure 5) demonstrated the difference in photobleaching rates. We found (by linear regression analysis) that the photobleaching rate of the Fast Red TR azo dye adduct was more than three times greater than that of the ELF-97 precipitate.



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Figure 5. Fluorescence signal photostability comparison of the ELF-97 alcohol precipitate and the Fast Red TR azo dye adduct. Cryosections of the zebrafish kidney stained by the ELF-97 alcohol precipitate (filled boxes) and the Fast Red TR azo dye adduct (open circles) were illuminated for 90 sec with constant high-power UV light and images collected every 5 sec with a cooled CCD camera. The normalized data demonstrate the difference in photobleaching rates. By linear regression analysis, the photobleaching rate of the Fast Red TR azo dye adduct was found to be 9.4 x 10-4 fluorescence units/sec, whereas the rate of the ELF-97 alcohol precipitate was 3.3 x 10-4 fluorescence units/sec.

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 (McComb et al. 1979 ; Wheater et al. 1987 ; Harris 1990 ). In the kidney, the ELF-97 signals were localized to tubules having a very prominent brush border of microvilli projecting into the lumen of the tubule (Figure 6B). In contrast, other tubules could be identified by Hoechst staining that completely lacked any ELF-97 precipitate (Figure 6B, arrow). This observation is in agreement with findings that distal convoluted tubules do not express alkaline phosphatase and possess no brush border (McComb et al. 1979 ; Wheater et al. 1987 ; Harris 1990 ).



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Figure 6. Endogenous alkaline phosphatase activity in intestine and kidney counterstained with Hoechst stain. Cryosections of zebrafish intestine (A) and kidney (B) stained the ELF-97 alcohol precipitate and counterstained with blue-fluorescent Hoechst 33342 nucleic acid stain. In the intestine (A), cells located in the invaginated mucosa at the bases of the villi (area between the arrows) were not labeled by the ELF-97 alcohol precipitate. In the kidney (B), tubules having a prominent brush border were labeled by the ELF-97 alcohol precipitate whereas other unlabeled tubules (arrow) stained only with the Hoechst dye. Bar = 25 µm.

Figure 7. Ciliary body of the eye labeled with Texas Red-X–wheat germ agglutinin, the ELF-97 alcohol precipitate, and Hoechst stain. Sections through the ciliary body of the zebrafish eye stained with the ELF-97 phosphate only (A) or after staining with Texas Red-labeled wheat germ agglutinin (B). Sections were subsequently counterstained with Hoechst nuclear stain. The ELF-97 alcohol signal appears brighter in B than in A due to an underlying low level of wheat germ agglutinin stain which makes for a slightly more yellow ELF-97 alcohol signal. Bar = 25 µm.

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 (Burstone 1960 , Burstone 1962 ). To assess the compatibility of the ELF-97 phosphate method with blocking reagents, we examined the quality of the ELF-97 alcohol precipitation when endogenous alkaline phosphatase detection was performed after a standard blocking procedure with 1% BSA followed by application of a labeled binding protein, Texas Red dye-labeled wheat germ agglutinin. We examined the ciliary body of the eye, because the staining for endogenous alkaline phosphatase activity without the wheat germ agglutinin application produced an extremely well-defined pattern of labeling, with a very fine ELF-97 alcohol precipitate evident on the surface of the cells (Figure 7A). We compared this labeling to that produced when the ELF-97 phosphatase substrate was applied after the application of wheat germ agglutinin (Figure 7B). In those samples, the red fluorescent wheat germ agglutinin conjugates could be seen simultaneously with the yellow-green fluorescence of the ELF-97 signal and the blue fluorescent Hoechst nuclear staining. The labeling by the ELF-97 alcohol precipitate was well localized, showing sharply defined cell membranes in both applications (compare Figure 7A and Figure 7B). Furthermore, the precipitate was very fine and did not become granular, as has been observed for some azo dye adducts (Burstone 1960 , Burstone 1962 ; Ziomek et al. 1990 ).

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 (Low and Saltiel 1988 ). We found that development of the cytoplasmic signal was dependent on treatment that permeabilized the cell. Staining of permeabilized cells by other methods also showed a weak internal signal (Figure 8B and Figure 8D, arrows). In contrast, no internal signal was apparent in cells stained with the Fast Blue BB azo dye adduct, although the cell surface was clearly labeled (Figure 8C). In all cases, labeling was blocked by incubation with levamisol (Figure 8E–8H). Cells that were not permeabilized produced no internal label, but only signals on the cell surface (Figure 8I). To verify that the cytoplasmic signal was endogenous alkaline phosphatase activity, we performed a co-localization study using a monoclonal antibody against alkaline phosphatase (RBM 211.13) (Turksen and Aubin 1991 ; Turksen et al. 1992 ). The antibody localized alkaline phosphatase to the cell surface and, after permeabilization, to an intracellular locale (Figure 9A, arrow, and 9B). No signal was produced in controls performed without the primary antibody (Figure 9C). We next asked if the intracellular protein recognized by the antibody co-localized with the activity detected with the ELF-97 substrate. Cells were first assayed with the antibody and then with the ELF-97 phosphate, and both fluorescence signals were viewed simultaneously. We found that the two signals corresponded. The yellow-green fluorescence of the ELF-97 precipitate (Figure 10A) and the red fluorescent label of the antibody (Figure 10B) were seen as yellow fluorescence when viewed simultaneously (Figure 10C). Furthermore, both signals were located outside of the nucleus, which was stained fluorescent blue by the Hoechst stain (Figure 10C). These results indicate that both the antibody and the ELF-97 substrate identified the same perinuclear endogenous alkaline phosphatase.



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Figure 8. Endogenous alkaline phosphatase activity in UMR-106 osteosarcoma cells. Cells were stained with the ELF-97 alcohol precipitate (A,I), Fast Red TR azo dye adduct (B), Fast Blue BB azo dye adduct (C), and BCIP/NBT (D). In permeabilized cells, a perinuclear region in the cytoplasm was labeled, in addition to the cell surface, by three of the methods (A,B,D, arrows). Controls (E–H) were performed with 5 mM levamisol. (I) Cells that were not permeabilized but were stained using the ELF-97 phosphate. Bar: AD,I = 25 µm; EH = 100 µm.



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Figure 9. Antibody localization of endogenous alkaline phosphatase in UMR-106 cells. Cells were labeled with a monoclonal antibody against alkaline phosphatase (RBM 213.11) and detected with Alexa 594 dye-labeled goat anti-mouse IgG F(ab')2 secondary antibody. In permeabilized cells (A), a perinuclear region in the cytoplasm was labeled (arrow) in addition to the cell surface. (B) Cells not permeabilized. (C) Controls performed without the primary antibody. Bar = 25 µm.

Figure 10. Localization of endogenous alkaline phosphatase in UMR-106 cells using both the anti-alkaline phosphatase antibody and the ELF-97 phosphatase substrate. Permeabilized cells were stained for endogenous alkaline phosphatase, first with the anti-alkaline phosphatase antibody and then with the ELF-97 phosphatase substrate. Cells were counterstained with Hoechst nuclear stain. The overlapping signals of the yellow-green fluorescence of the ELF-97 alcohol precipitate (A) and the red fluorescence of the antibody (B) become yellow when viewed simultaneously (C). Note correspondence of the perinuclear signals (C). Bar = 25 µm.


  Discussion
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Materials and Methods
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Discussion
Conclusions
Literature Cited

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 (Low and Saltiel 1988 ). The activity that we detected in the cytoplasm was sensitive to the inhibitor levamisol, suggesting that it is a bone/liver/kidney/placental-type alkaline phosphatase (Goldstein et al. 1980 ). A monoclonal antibody against alkaline phosphatase co-localized with the activity in permeabilized cells, refuting the possibility that the substrate is being cleaved by some other phosphatase. We think that it is possible that this activity could reflect the presence of enzyme that was internalized during membrane turnover, present in vesicular structures where a phosphatase acting at alkaline pH is required, or that it is in transit out of the cell.

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 (Larison et al. 1995 ; Paragas et al. 1997 ), we found that signal development can be monitored at the microscope with very little loss of fluorescence. Because the signal develops rapidly in comparison with other substrates, we found that staining must be stopped promptly once the desired level of labeling is achieved. Typical staining reactions required 30–90 sec for optimal signal development; the exact time requirement was dependent on the level of localized enzyme activity. Kidney tubules, for example, express high levels of alkaline phosphatase and therefore label very quickly. For these samples, we found that adjusting the substrate concentration to half of that recommended by the manufacturer helped to slow the reaction to a rate that enabled us to obtain the best results. We found that overlabeling tends to lead to the formation of large granular crystals and spurious crystals that compromise labeling resolution and specificity.

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. Burstone 1960 also commented on the granularity of certain azo dye adducts with respect to the lipid content of the tissue. In some cases, we have found that signal granularity can be improved by prewashes of the tissue with nonionic detergents or ethanol. Because the reaction is directly dependent on the level of active enzyme in the sample, signal intensity is also related to the techniques used in tissue preparation. Careful attention should be paid, in particular, to the use of fixation and embedding techniques that have been optimized for preservation of alkaline phosphatase activity (Burstone 1960 ). Some protocols may not accommodate the reagent as well as others. However, we found that significant deviation from the procedures recommended by the manufacturer (Meltzer et al. 1997 ) almost always leads to suboptimal results.

Finally, although several researchers have published on the use of standard fluorescein photographic filters and excitation sources with the ELF-97 phosphatase substrate (Jowett and Yan 1996 ; Pecorino et al. 1996 ; Meltzer et al. 1997 ), the precipitate is optimally excited at about 360 nm in the ultraviolet and emission is optimally detected at about 530 nm. Excitation at 488 nm is extremely suboptimal because there is very little absorbance of the precipitate at that wavelength (Singer et al. 1994 ).

Comments on Optimizing Fast Red TR Azo Dye Techniques
In contrast to the observations of previous researchers (Dolbeare et al. 1980 ; Raap 1986 ; Kamalia et al. 1992 ), we did not observe high background staining with the Fast Red TR azo dye adduct. One possible explanation for this discrepancy is that we monitored fluorescent signal development as it occurred, to prevent over staining. As discussed above, we have found that overstaining is the most frequent cause of high background signals, low staining specificity, and poor resolution. Although the Fast Red TR azo dye adduct is visible with both light and fluorescence microscopy, we found, in agreement with others (Ziomek et al. 1990 ), that its fluorescent signal has much higher sensitivity than its colored signal. In addition, samples that were labeled optimally for colorimetric detection were overlabeled for fluorescent detection, whereas samples that were optimally labeled for fluorescent detection were underlabeled for colorimetric detection. Therefore, in our comparisons, we found it useful to monitor signal development of this substrate using the fluorescence microscope. We also found that exposure of the Fast Red TR azo dye adduct to intense white light can also result in bleaching of the sample, so that it is not the case that the same samples can be optimally prepared for both visualization methods.


  Conclusions
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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 (Gonzalez-Gil et al. 1998 ) and in avian and mammalian cells (Telford et al. unpublished results). Furthermore, we have found that the ELF-97 alcohol precipitate is very stable at acidic pH, and our preliminary studies indicate that the substrate is useful for localizing acid phosphatase activity in cells (unpublished results). Therefore, although the use of the method requires considerable care and somewhat strict adherence to protocols, it can provide sensitive, high-resolution localization of endogenous phosphatase activity in tissues and cultured cells.


  Acknowledgments

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.


  Literature Cited
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Summary
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Materials and Methods
Results
Discussion
Conclusions
Literature Cited

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