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.     

A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies

Eight different previously described chromogen protocols were evaluated with respect to their sensitivity for the visualization of horseradish peroxidase (HRP) in a peroxidase-antiperoxidase (PAP) complex used with the unlabeled antibody method for immunohistochemistry. The protocols were evaluated in a test system that involved the demonstration of immunoreactive neurofilaments (NF) or glial filaments (GF) in paraffin-embedded sections of rat cerebellum using anti-NF or anti-GF monoclonal antibodies (MA). The chromogens included: amino-ethylcarbazole (AEC), diaminobenzidine (DAB), O-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and tetramethylbenzidine (TMB). The incubation medium using DAB as the chromogen was employed at neutral pH, at pH 5.1, or at neutral pH with the addition of either cobalt chloride or imidazole to intensify the reaction product. The relative sensitivity of the chromogen protocols was quantitated by comparing the dilution of the anti-NF or anti-GF MA at which NF or GF immunoreactivity was extinguished using each protocol. The results obtained with both the anti-NF and anti-GF MA indicated that DAB with imidazole was the most sensitive chromogen protocol.     

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

Summary New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca-and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H₂O₂ which decomposes at 55°–60° C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H₂O₂ mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H₂O₂-DAB and especially the H₂O₂-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.     

Menadiol diphosphate,a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

Summary Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce³⁺ ions and the DAB−Ni−H₂O₂ procedure yielded better results than Ca²⁺ ions in the Co−(NH₄)₂S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce³⁺ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT. Supported by the Alexander von Humboldt-Stiftung and the Deutsche Forschungsgemeinschaft (Sfb 174)     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

Histochemical phenotypes of von Ebner's gland of ferret and their functional implications

Von Ebner's gland of ferret was examined by means of light microscopy, protein, mucosubstance and enzyme histochemistry, and neurohistology. Acinar cells were replete with granules containing neutral mucosubstances and disulphides, and showed strong diffuse acid phosphatase activity and weak granular staining for peroxidase. Staining for cytochrome oxidase, succinate dehydrogenase, and NADH and NAD(P)H dehydrogenases was also seen. Basolateral plasmalemma of acinar cells showed weak, ouabain-sensitive Na⁺,K⁺-ATPase activity. Ductal cells were of a simple appearance, contained thiols and showed variable staining for acid phosphatase, dehydrogenases and cytochrome oxidase. Variable amounts of -glucuronidase reaction product were localized in the glandular parenchyma, being marked in atrophic areas. Prominent stellate myoepithelial cells embracing acini and also basal ductal cells were demonstrated by alkaline phosphatase. Thiamine pyrophosphatase reaction product was concentrated in blood vessels around parenchyma, with little Golgi-like staining in acinar cells. Acetylcholinesterase activity was associated with an extensive network of nerve fibres embracing parenchyma, whereas catecholamine fluorescence was not seen. The results suggest that the acini of von Ebner's gland of ferret synthesise neutral secretory glycoproteins and peroxidase. Water mobilization is inconspicuous. Lysosomal activities feature in the parenchyma, possibly a consequence of processing secretory products in acini, absorption in ducts and/or adaptation atrophy. The gland receives a rich cholinergic-type innervation, and has extensive myoepithelial and microvascularbreak networks.     

External Ni<Superscript>2+</Superscript> and ENaC in A6 Cells: Na<Superscript>+</Superscript> Current Stimulation by Competition at a Binding Site for Amiloride and Na<Superscript>+</Superscript>

In cultured A6 monolayers from distal Xenopus kidney, external Ni²⁺ stimulated active Na⁺ uptake via the epithelial Na⁺ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni²⁺ effect. Na⁺ current noise analysis was performed using the neutral Na⁺-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni²⁺ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni²⁺ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni²⁺ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na⁺—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni²⁺ exerted mutually exclusive analogous effects on amiloride kinetics. Na⁺ current kinetics revealed that Ni²⁺ prevents ENaC to be downregulated by self-inhibition. Co²⁺ behaved similarly to Ni²⁺, whereas Zn²⁺ did not. Attempts to disclose the chemical nature of the site reacting with Ni²⁺ suggested cysteine but not histidine as reaction partner.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

Non-radioactive in situ hybridization

Summary A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT+CIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetyl-aminofluorene (AAF)) were used. The advantages and dis-advantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.     

Comparison Between Two Chromogenic Substrates for Revealing an Immunoperoxidase Reaction of Human Metaphase Chromosomes

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.     

A Biolchemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: I. A COMPARISON OF DAB-PEROXIDASE AND GUAIACOL-PEROXIDASE WITH PARTICULAR EMPHASIS ON THE PROPERTIES OF CELL WALL ACTIVITY

Peroxidase activity in roots of Pisum sativum has been examinedusing both guaiacol and 3,3'-diaminobenzidine (DAB) as hydrogendonors. Biochemically, differences were observed between thetwo donors with respect to the pH optimum (6–9 and 4–0,respectively), and in response to added NaCl (guaiacol-peroxidasewas unaffected while the DAB-peroxidase was markedly inhibited).Both reactions showed highest specific activity in a high speedsupernatant fraction, and, of nine anionic bands demonstratedby gel electrophoresis with DAB, only six were visible withguaiacol. Histochemically, similar staining patterns were observedwith both donors. Cell wall fractions prepared by bead filtration contained 2%and 3.5% of the total peroxidase and acid phosphatase activitiesrespectively. 50% and 27% of these activities were ionicallybound, as indicated by salt treatment In addition, washing withsalt solutions produced a marked stimulation of peroxidase activityat high salt concentrations: this affect was not observed withthe supernatant peroxidase or with cell wall acid phosphatase.Possible functions of cell wall peroxidase are discussed     

Metalloenzyme inhibitor from kidney beans: partial purification and characterization

Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn²⁺. One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn²⁺. The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺. The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn²⁺ from the enzymes.     

Histamine content of peritoneal and tissue mast cells of growing rats

Summary p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation     

Novel salt-resistant alkaline phosphatase from eggs of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

It was shown that the alkaline phosphatase (AP) isolated from eggs of the sea urchin Strongylocentrotus intermedius was a homodimer with a molecular mass of 150 kDa and that it exhibited maximal activity at a рН of 8.1–8.5 and a temperature of 45°С. Mg²⁺, Ca²⁺, and Mn²⁺ ions, as well as DTT, are activators of AP activity, while Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, and Ni²⁺ ions, as well as EDTA, N-EMI, and p-CMB, inhibit its activity. The enzyme demonstrates unique salt resistance and can hydrolyze the substrate in seawater. It was shown by fluorescence methods and circular dichroism that an increase of the NaCl concentration above 1.0М caused noticeable changes in the secondary and tertiary structures of the protein and a decrease in enzyme activity. Analysis of the molecular masses of tryptic peptides conducted by mass spectrometry with the MASCOT program (which is based on the NCBI and SWISS-PROT databases) did not allow identification of the investigated protein. The uniqueness of the source and of the properties of the investigated AP, which were not characteristic for nonspecific AP, as well as the difficulties in primary structure identification with the fingerprinting technique, suggested that this enzyme was a nontypical AP with a novel structure.     

Comparison between two chromogenic substrates for revealing an immunoperoxidase reaction of human metaphase chromosomes

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.     

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.     

Copper-H2O2 oxidation strikingly improves silver intensification of the nickel-diaminobenzidine (Ni-DAB) end-product of the peroxidase reaction

We propose an improved silver procedure for intensification of peroxidase staining. It utilizes the high argyrophilia of polymerized Ni-DAB as a chromogen of peroxidase histochemistry, and the capacity of Cu++-catalyzed H2O2 oxidation to suppress tissue argyrophilia without influencing the argyrophilia of the polymerized Ni-DAB. This procedure is much more effective than any previously proposed intensification technique. When used in somatostatin histochemistry, it reveals perikarya, fibers, and nerve terminals in locations at which they have never been detected in preparations where only the DAB polymer was silver-intensified.     

Alkaline phosphatase activity in the brain of the American cockroach Periplaneta americana L

Summary The supra- and suboesophageal ganglia of the American cockroach contain material which catalyses the alkaline hydrolysis (pH 9.5) of 5-bromo-4-chloro-3-indolyl phosphate in the presence of Nitro blue tetrazolium. Histochemical studies on unfixed cryostat sections indicate that this type of alkaline phosphatase is restricted to discrete regions in the cockroach brain. Highest enzyme activity is encountered in the mushroom bodies, central body, antennal glomeruli and specific parts of some distinct neural connections including the optic nerve, antennal nerve, circumoesophageal connectives and nerves leaving the suboesophageal ganglion. Tissue fixation by use of formaldehyde-type fixatives, as well as routine paraffin-embedding, completely destroy all histochemically detectable enzyme activity.Native polyacrylamide gradient electrophoresis suggests that the alkaline phosphatase activity is present as multiple isozymic forms, which show up in the 120–130 kD range of standard proteins. Enzyme activity becomes undetectable after fixation (trichloroacetic acid, formaldehyde containing fixatives) of electrophoretically separated native proteins, as well as after electrophoresis in denaturing conditions (SDS and -mercapto-ethanol, boiling). However, the enzyme activity remains virtually unaffected after storage of the sample for prolonged periods at –20 to –80°C.