Diaminobenzidine Reaction Research Articles

Autophagy of peroxisomes in hepatic parenchymal cells.

This study was carried out to elucidate the mechanism of an autophagy of peroxisomes seen in hepatic parenchymal cells in the CPIB-treated rat liver followed by partial hepatectomy or by glucagon administration. The catalase activity demonstrated cytochemically by the diaminobenzidine reaction was used as a marker for peroxisomes and the acid phosphatase activity demonstrated by the lead method for lysosomes.The large size and number of peroxisomes induced by the CPIB administration in the rat hepatic parenchymal cell showed a gradual decrease in number after partial hepatectomy with concomitant increase in the number of lysosomes of various sizes including autophagolysosomes. Autophagoly-sosome formation involving peroxisomes was also evident in the hepatic parenchymal cells in the CPIB-treated and glucagon-administered rat liver. Furthermore, participation of the lysosomal wrapping mechanism in autophagocytosis of peroxisomes was frequently observed.It was concluded in the present investigation that the lysosome participates in the catabolism of peroxisomes observed in the CPIB-treated rat liver followed by partial hepatectomy or glucagon administration.

Hypothalamic, other diencephalic, and telencephalic neurons that project to the dorsal midbrain.

Neurons in the hypothalamus, other diencephalic regions, and the telencephalon which project to the mesencephalic central gray (CG) and the region lateral to it were demonstrated, in the rat, by the horseradish peroxidase retrograde neuroanatomical tracing method with diaminobenzidine and tetramethyl benzidine visualization reactions. The greatest concentrations of neurons that project to the dorsal mesencephalon were found in the ventromedial nucleus, particularly the anterior and ventrolateral subdivisions, in the dorsal premammillary nucleus, and in the zona incerta. Neurons that project to or lateral to the CG were also found in the laterocaudal hypothalamus, the dorsomedial hypothalamus, regions of the anterior hypothalamic area, specific areas of the cerebral cortex (32, 29, 8, 8A, 13, 14), and the central nucleus of the amygdala. Some neurons that project were also found in the preoptic area, septum, bed nucleus of the stria terminals, and the habenula. More neurons in the mediocaudal quadrant of the hypothalamus project to the mesencephalon than do those in laterocaudal, mediorostral, or laterorostral quadrants. More neurons in the medial than the lateral half, and more in the caudal than the rostral half of the hypothalamus project to the mesencephalon. More neurons project to the central gray, or the region lateral to it, at the levels of the superior colliculus, or intercollicular region, than at the level of the inferior colliculus. These descending connections to the midbrain, particularly from the hypothalamus and zona incerta, are probably components of neural networks that regulate nociception, certain neuroendocrine functions, sexual and other behaviors, and certain autonomic functions.

Ultrastructural localization of oxidative and peroxidative activities in a carrot suspension cell culture

Oxidative and peroxidative activities were localized at the ultrastructural level in suspension cells of an anthocyanin-producing strain of carrot after treatment with dihydroxyphenylalanine (DOPA) and diaminobenzidine (DAB). In DOPA-treated cells a reaction ascribed to polyphenoloxidase (PPO) occurred in the thylakoids of plastids. After DAB treatment at pH 9.0 reactions occurred in microbodies and plastid thylakoids; after treatment at pH 6.8 additional reactions occurred in the mitochondrial cristae and cytoplasmic ground substance. No reaction occurred in the cell walls at either pH. A reaction could not be unequivocally detected in the vacuoles because of the natural occurrence of osmiophilic material. Application of peroxidase and PPO inhibitors indicated that four distinct systems were involved in the DAB reactions: catalase was correlated with the reaction in the microbodies, peroxidase with the reaction in the cytoplasmic ground substance, cytochromes with the mitochondrial reaction, and PPO with the reaction in the thylakoids of the plastids.

Unlabeled antibody peroxidase-antiperoxidase method combined with direct immunofluorescence.

Paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method and direct immunofluorescence (DIF) differentiated distinctly between gastrin- and somatostatin-producing cells in the human gastric antrum. Similar paired staining of complexed lambda and alpha chains in immunoglobulin (Ig)A myeloma cells, of kappa and free J chains in IgG myeloma cells, and of secretory IgA and its epithelial transport protein, the free secretory component (SC), in colonic crypt cells, demonstrated that PAP staining inhibits subsequent DIF staining of an antigenic determinant present on the same molecule as the antigen revealed by the brown color of diaminobenzidine (DAB) or present or an unassociated molecule in the same cell. A quenching effect of the DAB reaction product was noted for both fluorescein (green) and rhodamine (red) emissions. In addition, a blocking effect of the DAB deposits has been demonstrated and is assumed to be the principal methodological basis for the paired PAP-DIF staining approach omitting intermediate antibody elution, as well as for the more time-consuming sequential PAP staining with DAB substrate for the first and 4-chloro-1-naphthol (CN) for the second antigen. The quenching and blocking effects limit in practice the paired PAP-DIF method to the localization of antigens present in separate cells.

Input and output synapses on identified motor neurones of a locust revealed by the intracellular injection of horseradish peroxidase.

Physiologically characterised motor neurones in the thoracic ganglia of the locus were injected with horseradish peroxidase in order that the spatial relationship between their input and output synapses could be observed with the electron microscope. A modification in the development procedure for the peroxidase ensured that the internal fine structure of the stained neurones was not obscured by the diaminobenzidine reaction product. Input and output synapses may occur within 1 micrometer of each other on the neuropilar processes of the motor neurones. This supports physiological evidence that motor neurones may be involved in local circuit interactions within the thoracic ganglia.

Cytochemical studies on peroxisomes in rat and guinea pig myocardium.

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.

The specific binding of parotin on duct cells of human parotid gland

The relationship between parotin and human parotid gland was investigated using indirect technique of enzyme antibody method. Normal rabbit IgG has non-specific affinity to duct cells of human parotid gland, but it was suppressed by adding bovine serum albumin. Anti-parotin rabbit IgG has no reaction products on the sections of adult human parotid gland. When the sections, initially incubated with parotin solution in PBS, were treated with anti-parotin rabbit IgG containing bovine serum albumin, and then HRPO-labeled anti-rabbit gamma globulin antibodies (swine IgG), intercalated and striated ducts excepting excretory ducts were stained with diaminobenzidine reaction, but also acinar cells did not. These results indicate that parotin is bound specifically on duct cells of human parotid gland.

Microbodies in the Brown Rot Fungus Poria contigua

Summary Using the diaminobenzidine (DAB) reaction catalase activity could be demonstrated cytochemically in cytoplasmic structures of Poria contigua bearing the general ultrastructural features of microbodies. These kidney-shaped compartments were associated with a densely packed coherent membrane system. When H 2 O 2 was replaced by methanol the microbodies were stained indicating that alcohol oxidase generated the H 2 O 2 for the peroxidative conversion of DAB by catalase. Based on these results we assume that catalase and alcohol oxidase are located in microbodies of this fungus as found for methanol-utilizing yeasts.

Ovoperoxidase activity in ionophore treated mouse eggs. I. Electron microscopic localization

AbstractA mammalian ovoperoxiadase activity has been detected in ionophore activated mouse eggs. The peroxidase activity was demonstrated at the electron microscopic level using the 3,3′‐diaminobenzidine (DAB) histochemical method. A positive DAB reaction was detected in a portion of the intact cortical granules of untreated or DMSO treated control eggs. In the ionophore activated eggs, the DAB reaction product was routinely detected by electron microscopy, predominantly on the cell surface, that is on the zona pellucida, in the perivitelline space, and in association with the cortical granule exudates. Furthermore, the peroxidase inhibitors phenylhydrazine and sodium sulfite prevented DAB staining in ionophore activated oocytes. These results indicate the presence of an ovoperoxidase, possibly of cortical granule origin, on the surface of activated mammalian eggs, detectable by histochemical means.

Temporal specificity of hemoglobin synthesis in the fat body of Chironomus thummi during development

AbstractHemoglobin (Hb) synthesis in the subepidermal fat body cell during the development of Chironomus thummi was investigated by using positive diamino‐benzidine (DAB) reaction as an indicator for the presence of Hb. The localization of DAB reaction products in the ER and Golgi cisternae is stage‐specific. Active synthesis of Hb, as indicated by heavy deposits of reactive products, is interrupted during third‐fourth larval ecdysis, resumes about the fourth day of the fourth instar, continues throughout the fourth instar and stops again shortly before the transition from the pharate pupa to the pharate adult at which time the fat body shows numerous autophagic vacuoles. These results support the concept of the existence of specific switches in the production, release and degradation of Hb present in Chironomus.

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.

Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.

The comparative effectiveness of the ‘brown and blue reactions’ for tracing neuronal processes of cells injected intracellularly with horseradish peroxidase

The comparative advantages and disadvantages of diaminobenzidine (DAB) and benzidine di-HCl (BDHC) procedures for tracing processes of cells injected intracellularly with horseradish peroxidase (HRP) have been studied. The DAB reaction gives a good definition of membranes and appears more suitable for visualizing the fine structure of dendrites. The BDHC procedure, being more sensitive, induces crystal formation and heavily filled processes appear frosted. This last procedure is, however, much more appropriate for tracing the axonal system of injected cells.

Fine structure and cytochemical properties of tobacco leaf protoplasts and comparison with the source tissue

Protoplasts isolated from tobacco leaves showed the development of crystalline bodies in the chloroplasts and osmiophilic droplets in the chloroplasts and cytoplasm. Microbodies stained for catalase with the diaminobenzidine (DAB) reaction although, in the absence of H2O2, mitochondrial cristae also showed a dense reaction. Similar changes and DAB staining were observed in intact leaf cells aged in media similar to that used for protoplast isolation. The cell wall and plasma membrane of fresh leaf cells stained inconsistently with silicotungstic acid (STA). In comparison, the plasma membrane of freshly isolated protoplasts stained very poorly with STA although more intense staining was found in aged protoplasts. The surface of protoplasts showed heavy staining when incubated in lanthanum nitrate or cationized ferritin though not with ruthenium red. The potential of these surface stains as plasma membrane markers in isolated membrane fractions is discussed.

Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.

Diaminobenzidine reactions in control and treated Amoeba proteus.

Cytochrome oxidase activity was demonstrated in Amoeba proteus by diaminobenzidine (DAB) cytochemistry. Deposition of the reaction product occurred on the inner mitochondrial membranes and the cristae. The reaction was abolished by cyanide incubations. Positive reactions were produced with both unfixed and fixed cells: although staining potential was destroyed by any prefixatives which included glutaraldehyde. Cells prefixed with 4% formaldehyde, to raise structural preservation, retained staining ability. Amoebae subjected to prolonged anaerobiosis or to treatment with the carcinogen N-methyl-N-nitrosourethane (MNU) displayed a reduction in DAB reactivity. A positive reaction was only produced in incubations of unfixed cells and even in these the intensity of cristal staining was depleted. The possible use of DAB reactions where lesions in mitochondrial functioning have occurred is considered.

Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland.

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3′-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.

Ultrastructural localization of catalase and L-alpha-hydroxy acid oxidase in microperoxisomes of Hydra.

The ultrastructural localization of catalase and L-alpha-hydroxy acid oxidase (LalphaHAO) was studied in two species of Hydra. Diaminobenzidine reaction product of catalase activity was present in small round or elongated bodies resembling microperoxisomes in the epitheliomuscular, digestive and gland cells. They were closely related to the endoplasmic reticulum, and were often found in proximity to deposits of lipid and glycogen. Reaction product of LalphaHAO activity was also associated with the microperoxisomes. With rapidly oxidized substrates, such as L-lactic acid, reaction product diffused into the cytoplasm around the microperoxisomes. With slowly oxidized substrates, such as DL-alpha-hydroxyisovaleric acid, reaction product was restricted to the matrix of the microperoxisomes. No reaction product was present in the microperoxisomes in the absence of substrate or with D-lactic acid. The rate of substrate oxidation measured biochemically roughly paralleled the amount of cytochemical reaction product deposited with different substrates. Microperoxisome-like bodies reactive for LalphaHAO were also found in the epidermal cnidoblasts; however, catalase could not be demonstrated in them. This study provides the first cytochemical evidence for the presence of an H2O2-producing oxidase in microperoxisomes.

Feeding mechanisms in extracellular Babesia microti and Plasmodium lophurae.

Although large hemoglobin inclusions are observed in intraerythrocytic Babesia microti parasites, they are absent from parasites freed of hamster red cells by immune lysis with anti-hamster erythrocyte serum. Babesia microti has no cytostome. This parasite, therefore, does not appear to feed by phagocytosis of large boluses of hemoglobin, as does Plasmodium. To determine whether Babesia can pinocytose protein, free parasites were fed ferritin in an in vitro system. Ferritin was taken up from the entire cell surface into narrow channels within 15 min at 37 C. Only merozoites, with their pellicular complex, failed to take up the protein. By 60 min, the ferritin was highly concentrated in many channels and vesicles, which formed interconnecting stacks. The ferritin-containing channels became associated with membrane whorls of the multimembranous structure. Membrane whorls were also observed in the process of extrusion in samples incubated for longer times. These events may represent steps in the digestion and excretion of the pinocytosed protein. Empty channels formed when Babesia was fed albumin. The diaminobenzidine reaction for hemoprotein was positive for the channels in both free and intraerythrocytic babesias. The staining reaction was completely inhibited by cyanide, but not at all by aminotriazole. These results further suggest that Babesia pinocytoses hemoglobin in vivo. Plasmodium lophurae parasites freed of red cells by immune lysis are surrounded by 2 membranes and apparently can ingest ferritin only through the cytostome. Extracellular cytostomal feeding involves both membranes, as it does in vivo. Ferritin was found in food vacuoles, some of which contained hemoglobin ingested before parasite isolation, connected to or near the cytostome. In both Plasmodium and Babesia low temperature inhibited ferritin uptake.