Light microscopical visualization of phosphatase activities demonstrated with the cerium method in resin sections of the kidney with the microwave oven

The light microscopically invisible reaction product cerium phosphate in resin sections of rat kidney, that had been incubated for the demonstration of phosphatase activities before embedding, was converted into a visible reaction product by incubation for 10 min at 80 degrees C in alkaline lead citrate in a microwave oven. This method offers the possibility to study phosphatase activities with the cerium method in semithin Epon sections. Furthermore it is a suitable method to select areas with phosphatase activity to be studied with the electron microscope.     

Ultrastructural localization of several phosphatases with cerium

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.     

Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.     

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes

Summary New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5–60 min at 37° C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2–9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure. However, in contrast to the other methods especially the cerium citrate procedure yielded a more precisely localized and more stable reaction product, can be used with all available alkaline phosphatase substrates including those up till now less suitable or unsuitable for light microscopic alkaline phosphatase histochemistry.     

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.     

Specific visualization of precipitated cerium by energy-filtered transmission electron microscopy for detection of alkaline phosphatase in immunoenzymatic double labeling of tyrosine hydroxylase and serotonin in the rat olfactory bulb

To understand in detail the functional morphology of neuronal circuits it is important to identify at the ultrastructural level the incoming axon, its target neuron, and members of the signaling cascades involved. This, however, represents a formidable task, requiring highly sophisticated electron microscopic multiple-labeling techniques. To extend available double-labeling procedures such as combinations of immunogold and peroxidase methods, an additional, gold- and peroxidase-independent procedure would represent a considerable advantage. The present investigation therefore aimed to use alkaline phosphatase as the immunoenzymatic label at the electron microscopic level via cerium phosphate precipitates. To our surprise we found that available techniques, which are well established for the visualization of endogenous enzymes in sections from various tissues, are not suitable for application to immunocytochemistry. Careful characterization of the individual reaction conditions, however, resulted in an optimized procedure with largely increased sensitivity. The novel technique yields cerium-containing precipitates which are massive enough to allow the detection of the immunoenzymatic reaction product in the electron microscope. Using the rat olfactory bulb as the model system we showed further that our technique allows the combination with the peroxidase/diaminobenzidine system for ultrastructural double labeling. For this purpose, the alkaline phosphatase product is identified by its cerium content via energy-filtered transmission electron microscopy and thereby differentiated from cerium-free peroxidase-derived precipitates. Doing so, we found that ascending serotoninergic fibers do not establish synapses with dopaminergic periglomerular cells in the rat olfactory bulb.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

Summary The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.     

New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Comparison with the lead technique

The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogeneous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Improved light microscopic demonstration of D-amino acid oxidase activity in cryotome sections using cerium ions as capturing and amplifying agent--the Ce/Ce-H2O2-DAB procedure.

The light microscopical demonstration of D-amino acid oxidase (AAOX) activity with cerium (Ce III) as the capturing agent was improved. The incubation medium was stabilized by the employment of triethanolamine and detrane complexed cerium. A considerable increase in intensity of the reaction was accomplished by treatment of the AAOX-incubated sections with Ce III which reacted with the primary reaction product Ce IV-perhydroxide to form Ce IV-hydroxide. In this way the primary reaction product was reduced and enlarged concomitantly. The Ce IV-hydroxide was converted into Ce IV-perhydroxide by H2O2, which was visualized by blue-black stained Ni-DAB complexes. Thus, Ce III is used as capturing agent as well as amplifier (Ce/Ce-H2O2-DAB method). The primary reaction product Ce III-phosphate formed by coreacting phosphatases was selectively extracted by citrate containing glycine-NaOH buffer while Ce IV-perhydroxide remained in the sections. In model experiments it was proven that the perhydroxide groups in the Ce IV-perhydroxide compound initiate predominantly the DAB polymerization while the contribution of Ce III and Ce IV is small.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Multivesicular bodies isolated from rat hepatocytes. Cytochemical evidence for transformation into secondary lysosomes by fusion with primary lysosomes

Plasma lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ. Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70-120 nm in diameter, which stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive arc or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, "intact" Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatocytes in situ. These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartments.     

New,improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

Summary New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Extinction coefficient of polymerized diaminobenzidine complexed with cobalt as final reaction product of histochemical oxidase reactions

The extinction coefficient is essential for the conversion of cytophotometric (mean integrated) absorbance values into absolute units of enzyme activity, for instance expressed in terms of moles of substrate converted per unit time and per unit wet weight of tissue. The extinction coefficient of polymerized diaminobenzidine (polyDAB) complexed with cobalt as the final reaction product of oxidase reactions was estimated at 575 nm by comparison of the amounts of final reaction products formed after incubation of serial unfixed cryostat sections of rat kidney to demonstrate D-amino acid oxidase activity with either the tetrazolium salt method or the cerium-DAB-cobalt-hydrogen peroxide method. Both procedures resulted in similar localization patterns of final reaction product in a granular form in epithelial cells of proximal tubules in rat kidney. The granules were peroxisomes. Linear relationships were found for both methods between the specific amounts of final reaction product generated by D-amino acid oxidase activity and incubation time. The cerium salt method gave rise to 7.4 times higher absorbance values of final reaction product generated per unit time and per unit wet weight of tissue than the tetrazolium salt procedure. The extinction coefficient of tetranitro BT-formazan is 19 000 at 557 nm. Therefore, the cytophotometric extinction coefficient of the poly DAB-cobalt complex as final reaction product of oxidase reactions was established to be 140 000.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

Summary The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogenous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Photographic microdensitometry for evaluation of acid phosphatase activity at the electron microscope level

Summary The dry mass of reaction products in ultrathin sections was determined using electron micrographs of polystyrene spheres of known weight deposited on Formvar membranes and evaluating the negatives photometrically. This method was applied to the quantification of the final reaction product of the acid phosphatase reaction in a model system in which enzyme was incorporated in gelatin. The enzyme activity was demonstrated by the lead precipitation method and quantified by direct microphotometry at the light microscope level. Models were then embedded and sectioned for electron microscopy. Microphotometric values afforded by the electron negatives were in linear correlation with incubation times and enzyme concentration. Section thickness and its possible variations due to deformation or contamination under the electron beam were also evaluated. Measurements of lysosomal acid phosphatase activity in rat kidney sections served to illustrate the application of the technique.