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.     

Localization of acid phosphatase in the differentiating root meristem

The localization of acid phosphatase was studied by Gomori’s newer technique and by azo-coupling methods (α-naphthyl phosphate + fast red ITR or fast garnet GBC; AS or AS D phosphate + fast blue B or fast red violet LB) in the root tips ofVicia faba L. on paraffin sections (fixation with Wolman’s acidified ethanol) and on frozen sections (fixation with Baker’s calcium formol). Analogous results were obtained on the material treated in various ways and using different methods. In the broad bean, the reaction is most intense in the cap. In the meristematic zone, the primary core is more intensely stained than the ground parenchyma of the central cylinder. The phloem and xylem poles are usually strongly positive. Using both types of methods on Wolman fixed paraffin embedded material, essentially the same localization of acid phosphatase was found in the root tips ofRicinus communis L.,Lupinus luteus L.,Sinapis alba L.,Allium cepa L. as in the broad bean. InZea mays L. the rhizodermis and the hypodermic layers of the primary core were found to be most active. On sections of Wolman fixed paraffin embedded broad bean the most intense reaction was observed at pH 4.2–4.8. In the same material, both the azo-coupling and the Gomori reaction is inhibited by 10⁼² M NaF, but 10^?2 M tartaric acid only inhibits the Gomori reaction.     

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

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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tetrazolium method for non-specific alkaline phosphatase

Summary A technique for the histochemical demonstration of non-specific alkaline phosphatase was developed using a medium containing indoxyl phosphate and a tetrazolium salt, Nitro B.T. The tetrazolium salt was reduced to diformazan by the hydrogen ions released by the formation of either indigo or indigo white by reaction of the enzyme on the indoxyl phosphate.The localization in the organs investigated was similar to that obtained by the standard azo dye and lead techniques.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Effect of Phosphorus Nutrition on the Cellular Distribution of Acid Phosphatase in the Leaves of Lycopersicon esculentum L.

A histochemical study using light microscopy has been made ofthe distribution of acid phosphatase (EC 3.1.3.2 [EC] ) activity intransverse sections of fully expanded leaves of Lycopersiconesculentum grown in phosphate-deficient or sufficient media.Leaf tissues were prepared by two methods and were embeddedin paraffin wax. The location of acid phosphatase activity inleaf sections was determined by trapping orthophosphate releasedfrom p-nitrophenyl phosphate with lead acetate and subsequentlyconverting the lead phosphate to optically dense lead sulphide.In leaf sections from control tissue lead sulphide depositswere larpely confined to the spongy mesophyll cells. Whereasthe staining of the palisade cells was limited and of a granularnature, the staining of the spongy mesophyll cells was heavierand coincident with the outline of the individual cells. Moreover,the minor veins were more heavily stained than the surroundingmesophyll cells. Sections of phosphorus-deficient tissues wereheavily stained in both the palisade and spongy mesophyll layersand heavy deposits of lead sulphide were present in the regionsof the minor veins. It is suggested that the enhanced acid phosphataseactivity of the mesophyll cells in fully expanded leaves couldbe involved in the remobilization of phosphate within phosphorus-deficientplants, or be part of a phosphate transporting system, concentratingthe intracellular phosphate from the limiting supply in thesolution bathing the mesophyll cells. Lycopersicon esculentum L., tomato, acid phosphatase, phosphorus nutrition     

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. II. Effect of the composition of the incubation medium on the trapping of phosphate ions in acid phosphatase cytochemistry

Synopsis A model system developed for the study of the dynamics of capture reactions for diffusable compounds in cytochemistry served as a basis for the experiments reported in the present paper. The model was used to study the effect of the composition of the cytochemical medium on the trapping of phosphate ions by lead (II) ions in acid phosphatase cytochemistry. In this system a phosphate-containing solution and a lead-containing solution (cytochemical medium) are pumped along opposite sides of a polyacrylamide film. The phosphate concentration at which measurable precipitation starts in the film (critical phosphate concentration) was taken as a measure of the trapping efficiency of the cytochemical medium. The addition of -glycerophosphate and cytidine-5-monophosphate to a buffered lead-containing solution resulted in a higher critical phosphate concentration. Both substrates had an effect on the crystal form of lead phosphate. The addition of chloride ions and acetone, as well as decreasing the molarity of the acetate buffer of the cytochemical medium, were found to lower the critical phosphate concentration, whereas the addition of fluoride ions, glucose, and sucrose had no effect. From the effect of variations in the composition of the cytochemical medium on the trapping efficiency and the turnover number of acid phosphatase in the medium, it was possible to predict which cytochemical medium would be the most suitable for the demonstration of acid phosphatase activity in guinea-pig peritoneal exudate cells. The results were in accordance with the localization of acid phosphatase activity: the higher the trapping efficiency and the turnover number, the higher the amount of precipitate and the number of positive enzymatic sites. In this way an improved cytochemical medium for acid phosphatase was developed.     

Histochemical Demonstration of Acid Phosphatase in Hard Tissues

The action of the following decalcifying solutions for the demonstration of acid phosphatase has been studied: buffer solution acetic-acetate 0.05 M, pH 5; 2, 5, 10, 20, and 50% formic acid and 20% sodium citrate in equal parts (pH 2.6, 3, 3.8, 4.2, and 5); 0.5 M citric acid-NaOH, pH 4.2; Versene solution, 5%, pH 7. A comparative study of fixatives has been made also (neutral formalin, 10-20%; formalin-chloral hydrate (Fishman), acetone and 80% alcohol). The best results were obtained with fixation at 4°C in 10-20% neutral formalin or formalin-chloral hydrate, for a period of 24 hr, and decalcification with 20% sodium citrate, 5% formic acid, in equal parts, pH 4.2, which can act on both specimens or sections for a period up to 2 wk with very little loss of enzyme. It is not necessary to reactivate the enzyme after decalcification; frozen sections should be used and should be washed in distilled water before proceeding with the demonstration of the enzyme (Gomori's method or azo dye coupling). Other fixatives (acetone and alcohol) and paraffin embedding produce a greater loss in enzymes and very irregular results.     

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

Summary 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° C in alka-line 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.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

The Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

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.     

A Chemical and Histochemical study of the Technic for acid Phosphatase

A chemical and histochemical study of Gomori's acid phosphatase technic showed that the causes of its unreliability were: (1) that fixation and other steps of histological procedure inactivate the enzyme to a great extent; (2) the enzyme may diffuse, as demonstrated in frozen sections of acetone-fixed material; and (3) some absorption of lead by the sections takes place. Much of this unreliability is avoided, however, by maintaining as low a temperature as possible during fixation and dehydration, with exposure to the temperature of the paraffin oven for the shortest possible length of time. The relative insolubility and thermo-stability of the enzyme, moreover, indicate the possibility of devising a more satisfactory technic in the future.     

Selective visualization of human dendritic reticulum cells in reactive lymphoid follicles by the immunohistochemical demonstration of the subunit A of factor XIII (F-XIIIa)

Immunohistochemical techniques have been used to localize clotting factor XIII subunit A in human reactive lymphoid follicles. The follicular dendritic reticulum cells (DRCs) were identified by the monoclonal antibodies R4/23 and OKB-7 as well as by their 5'-nucleotidase positivity. Follicular histiocytic reticulum cells (HRCs) were demonstrated by their acid phosphatase and non-specific esterase reactions. Capillaries were selectively visualized by adenosine triphosphatase. The immunohistochemical demonstration of F-XIIIa was preferably carried out in combination with one or two of the above marker techniques, on the same cryostat section. The subunit A of factor XIII is present in follicular DRCs. Their selective immunohistochemical demonstration with antibody against F-XIIIa requires formaldehyde fixation of cryostat sections. Similar fixation, however, is inappropriate for the demonstration of F-XIIIa reactivity of DRCs in paraffin sections. For this purpose, acetic acid-formalin fixation is useful. Follicular HRCs are consistently negative for F-XIIIa, contrary to the F-XIIIa positivity of sinusoidal and interfollicular HRCs. Developmental and functional implications of F-XIIIa reactivity in DRCs and HRCs are suggested.     

Enzyme Histochemistry on Paraffin Embedded Tissue Sections

To obtain diagnostic enzyme reactions in paraffin embedded tissue sections, we compared four fixatives (buffered formol sucrose, Baker's formol calcium, periodate lysin paraformaldehyde, and buffered formalin acetone) and subsequent acetone dehydration with or without graded concentrations of Triton X-100. Four spleens and 14 lymph nodes were tested for peroxidase, naphthol ASD chloroacetate esterase, acid phosphatase, alpha naphthyl acetate esterase, and alpha naphthyl butyrate. Best results were obtained by a processing method using buffered formalin acetone, Holt's gum sucrose, dehydration in acetone with 0.03% Triton X-100, and paraffin for embedding.     

Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections

Summary In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

Metachromatic Reaction of Pancreatic B Cells to Toluidine Blue; Influence of pH on Staining

The granules of islet B cells show an intense β metachromasia when paraffin sections of pancreas fixed in Bouin's fluid or formalin are dipped for 1 min in a 0.1% aqueous solution of toluidine blue O₂ buffered to pH 6.0 with acetate or phosphate. This reaction provides a quick method for surveying the condition of B cells in experimental work. A weak staining is observable at pH 4.5 and becomes distinct at pH 5.5-6.0. Oxidation of sections (0.25% KMnO₄ in 0.5% H₂SO₄, for 1 min, recommended) prior to staining intensifies the metachomatic reaction conspicuously. The metachromatic substance could not be demonstrated after fixation in either ethanol or acetone. It corresponds to the aldehyde fuchsin-positive and pseudoisocyanin-metachromatic substance in its occurrence and distribution in the B cells, as shown by different physiological states of various animals, including fasted and glucose-administered guinea pigs. It is thought to be topographically coincident but not necessarily identical to insulin.