A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

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)     

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus,Magnaporthe grisea

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

Uptake of Lucifer Yellow CH into plant-cell protoplasts: a quantitative assessment of fluid-phase endocytosis

The highly fluorescent dye Lucifer Yellow CH (LYCH), now in common use in microinjection studies, has been shown to enter the vacuole of a range of plant-cell protoplasts from the external medium. Uptake was quantified by lysing the protoplasts following incubation and determining the amount of LYCH incorporated by spectrofluorimetry. Uptake was biphasic with respect to both time and substrate concentration, enhanced at low pH and inhibited by low temperature and metabolic inhibitors. The kinetics of uptake showed several similarities with those reported for the fluid-phase endocytosis of LYCH in animal cells and yeast cells. A calculated membrane permeability coefficient for LYCH, based on the observed rates of uptake, was too high to be consistent with simple diffusion of the undissociated form of the molecule and inconsistent with the membrane-impermeant properties of the dye. The data are discussed in the light of the possibility of fluid-phase endocytosis versus active transmembrane transport.Abbreviations CCCP carbonyl cyanide M-chlorophenyl hydrazone - LYCH Lucifer Yellow CH     

Structure and function of the thecogen cell in contact chemosensitive sensilla of Periplaneta americana L. (Blattodea: Blattidae)

The functional morphology of the thecogen cell of the contact chemosensitive sensilla of the ventral sensory field on the maxillary palps of Periplaneta americana (Blattodea : Blattidae) was studied. There were electron-dense granules, which were examined using light and electron microscopy. Ultrastructural findings and acid phosphatase cytochemistry showed that these granules are lysosomes. The plasma membrane of the thecogen cell bordering the inner sensillum lymph also showed numerous coated pits. Intense fluid-phase endocytosis from the inner sensillum lymph into the thecogen cell was observed using Lucifer yellow as a fluorescent dye for infiltration. The endocytosed material is transported proximally and seems to be digested via the endosome-lysosome pathway. Lysosomes and endocytosis may serve the following functions: (1) the cleaning of the sensillum lymph from impurities entering via the tip porus; (2) the catabolic turnover during late embryonic development and before molting; (3) the continuous removal of stimulus molecules from the inner sensillum lymph after stimulation.     

Effects of prolonged exposure to ammonia on fluid-phase,receptor-mediated,and adsorptive (non specific) endocytosis in cultured neuroblastoma cells

Summary The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Light microscopic demonstration of myoid material in nuclei

Summary In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Intact barley (Hordeum vulgare L.) roots have been shown to take up the highly fluorescent dye Lucifer Yellow CH (LYCH) into their cell vacuoles. In the apical 1 cm of root tip, differentiating and dividing cells showed a prolific uptake of LYCH into their provacuoles. The LYCH was retained during fixation, apparently becoming bound to electron-dense material in the vacuoles. The dye freely entered the apoplast of roots in which the Casparian band was not developed, being taken up into the vacuoles of cells in both the cortex and stele. However, when LYCH was applied to a 1-cm zone approx. 6 cm behind the root tip the Casparian band on the radial walls of the endodermis completely prevented the dye from entering the cells of the stele, only the cell walls and vacuoles of the cortical cells taking up the dye. The inability of LYCH to cross the plasmalemma of the endodermal cells and enter the stele via the symplast substantiates previous claims that the dye is unable to cross the plasmalemma of plant cells. The results are discussed in the light of recent demonstrations that LYCH is a particularly effective marker for fluid-phase endocytosis in animal and yeast cells. A calculation of the energetic requirements for LYCH uptake into barley roots supports the contention that LYCH is taken up into the vacuoles of plant cells by fluid-phase endocytosis.Abbreviation LYCH Lucifer Yellow CH     

Immuno- and enzyme-histochemical detection of phosphoprotein phosphatase in rat epidermis

A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.     

Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices

The fluorescent dye Lucifer Yellow (LY) is a well-known and widely-used marker for fluid-phase endocytosis. In this paper, both light and electron microscopy revealed that LY was internalized into transition zone cells of the inner cortex of intact maize root apices. The internalized LY was localized within tubulo-vesicular compartments invaginating from the plasma membrane at actomyosin-enriched pit-fields and individual plasmodesmata, as well as within adjacent small peripheral vacuoles. The internalization of LY was blocked by pretreating the roots with the F-actin depolymerizing drug latrunculin B, but not with the F-actin stabilizer jasplakinolide. F-actin enriched plasmodesmata and pit-fields of the inner cortex also contain abundant plant-specific unconventional class VIII myosin(s). In addition, 2,3 butanedione monoxime, a general inhibitor of myosin ATPases, partially inhibited the uptake of LY into cells of the inner cortex. Conversely, loss of microtubules did not inhibit fluid-phase endocytosis of LY into these cells. In conclusion, specialized actin- and myosin VIII-enriched membrane domains perform a tissue-specific form of fluid-phase endocytosis in maize root apices. The possible physiological relevance of this process is discussed.     

A Stain for Bone—Illustrating Apposition and Absorption in Two Colors

Sections of either mineralized or demineralized tissues are incubated for 30 min at 37 C in a succinate-tetrazolium salt medium for the demonstration of succinic de-hydrogenase, and subsequently in a naphthol AS-MX phosphate and Red Violet LB salt mixture for 20 min at 25 C for the demonstration of alkaline phosphatase. Sections may be either mounted in a water-soluble medium, or dehydrated and mounted in a synthetic resin. Osteoclasts stain brown to purple; sites of osteoblastic activity, red.     

Fluorescence histochemical detection of hydrolases in tissue sections and culture cells

Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity and/or diffusion of the fluorescent tags. Satisfactory results are obtained for alkaline phosphatase, non-specific esterases and proteases with naphthol AS and 4-methoxy-2-naphthylamine coupled to nitrosalicylaldehyde. If, however, cultured monolayer cells are investigated, all synthetic substrates with fluorescent tags are suitable, including those that have so far only been used for biochemical hydrolase measurements. The fluorescent leaving groups are naphthol AS and its derivates, 4-methoxy-2-naphthylamine, aminomethylcoumarin, aminomethyltrifluoromethylcoumarin, methylumbelliferon, fluorescein and, with some limitations, also 1- and 2-naphthol. These fluorescence methods are more sensitive than the corresponding dye procedures. In addition, the fluorescence techniques allow the use of more synthetic substrates and therefore more information become available than with dye histochemistry about the enzymic properties of culture cells.     

Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches

Summary Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follice-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC₅(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction, is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC₅(3) fluorescence.Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smithet al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC₅(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.     

Detection of distinct endocytotic and phagocytotic activities in epithelial cells (pinacocytes) of freshwater sponges (Porifera, Spongillidae)

 Light and electron microscopical investigations using externally applied fluorescent and gold-labeled markers have revealed the existence of distinct endocytotic and phagocytotic activities in basal epithelial cells (pinacocytes) of the freshwater sponges Spongilla lacustris and Ephydatia es) of the f. The total rate of endocytotic membrane uptake, ascertained by the application of the cationic lipid probe TMA-DPH, was found to be 3.2% of the cell surface area/h. A typical fluid-phase endocytosis, demonstrated by the use of the water-soluble membrane-impermeable tracers BCECF-dextran and LY-CH, participates in endocytotic activity at a rate of 0.7% of the cell surface area/h and results in the formation of endosomes measuring 0.8–1 μm in diameter. Moreover, the application of labeled BSA succeeded in the detection of a receptor-mediated endocytosis amounting to a concentration-dependent uptake of 2.3–2.8% of the cell surface area/h. Coated pits and coated vesicles conveying the adsorbed BSA measure 0.3 μm in diameter and are covered on the cytoplasmic face with a clathrin-like protein (HC, 180 kDa; LC, 30 kDa). To facilitate phagocytotic activities, a series of fluorescent–labeled and chemically treated particles such as bacteria or latex beads have been successfully employed. Accordingly, the measured values of phagocytic membrane uptake between 1 and 8% of the cell surface area/h depend on the variety of size as well as the chemical nature of the different bioparticles and clearly point to phagocytosis as a key mechanism for providing freshwater sponges with nourishment. Accepted: 3 April 1997     

Cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase

Synopsis This paper describes how fluorogenic substrates derived from naphthol AS can be used for the microscopic demonstration and cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase in single living cells.A special study has been made of acid naphthol AS-BI phosphatase. However, the method can be extended to other hydrolytic enzymes. The method is sensitive and accurate because: quantification of very low enzyme activity is possible; the reaction kinetics can be evaluated with a good degree of precision inasmuch as the initial reaction velocity is derived over short times; there is an absence of distributional error; and the errors due to extra-cellular diffusion of the hydrolysed substrate, to photo-decomposition, and to autofluorescence can be contained within very narrow limits.The procedures for determining enzyme activity and reaction kinetics, and the instrumental characteristics and devices required for carrying out these measurements, are described. Some possible applications are indicated.     

Microbial decolorization of azo dyes by Proteus mirabilis

A bacterium identified as Proteus mirabilis was isolated from acclimated sludge from a dyeing wastewater treatment plant. This strain rapidly decolorized a deep red azo dye solution (RED RBN). Features of the decolorizing process related to biodegradation and biosorption were also studied. Although P. mirabilis displayed good growth in shake culture, color removal was best in anoxic static cultures. For color removal, the optimal pH and temperature were 6.5–7.5 and 30–35°C, respectively. The organism exhibited a remarkable color removal capability, even at a high concentration of azo dye. More than 95% of azo dye was reduced within 20 h at a dye concentration of 1.0 g L⁻¹. Decolorization appears to proceed primarily by enzymatic reduction associated with a minor portion, 13–17%, of biosorption to inactivated microbial cells. Received 06 January 1999/ Accepted in revised form 22 April 1999     

DIAZOPHTHALOCYANINS AS REAGENTS FOR FINE STRUCTURAL CYTOCHEMISTRY

This paper reports the synthesis of 14 diazophthalocyanins containing Mg, Cu, or Pb as the chelated metal. To assess the usefulness of these compounds for fine structural cytochemistry, the relative coupling rates with naphthols were tested as well as the solubility of the resulting azo dyes. Three of the diazotates were reacted with tissue proteins in aldehyde-fixed material, and the density increases thus produced were compared in the electron microscope with those produced by staining similarly fixed material with the phthalocyanin dye, Alcian Blue. Finally, one of the diazotates was used as a capture reagent for the demonstration of the sites of acid phosphatase activity with the electron microscope.     

A quantitative histochemical technique for the estimation of azo dye coupling reactions

Synopsis A quantitative histochemical method has been developed for the determination of the rate of formation of azo dye in an immediate coupling reaction for acid phosphatase. Predetermined areas of rat tail epidermis were continuously monitored by a photomultiplier. It is postulated that the rate of darkening is related to the rate of dye formation and that this in turn is dependent on the degree of enzyme activity in the area of tissue under observation. Regression coefficients were calculated for two different regions of rat tail epidermis. It was found that there was a significant difference between the transitional and lower epidermal zones:p<0.01 for 5 degrees of freedom.An aqueous model system showed that there was a relationship between the rate of dye formation and enzyme concentration. At present it is not certain whether this method can be used for the absolute estimation of enzyme in tissues, but it does appear to be satisfactory as a comparative technique.A slowing of the rate of reaction with time was clearly demonstrated and the greater the initial enzyme activity, the greater the rate of slowing. It is thought that this might be due to enzyme inhibition resulting from the azo dye formation.It is suggested that this method is relatively unaffected by variations in section thickness and this is a great advantage when cryostat sections are used. However, compression of tissue during sectioning does produce inaccuracies.