Diaminobenzidine Procedure Research Articles

Central projection of the peripheral cochlear nerve from each turn to the cochlear nuclei in the Mongolian gerbil.

The central projections of the peripheral cochlear nerve fiber from each turn to the cochlear nuclei (CN) in the mongolian gerbil were investigated using retrograde transportation of horseradish peroxidase (HRP). The organ of Corti and the osseous spiral lamina were scratched with an electrolytically-sharpened fine needle via a small hole at each turn of the cochlea. The cochlea was filled with a 30% horseradish peroxidase (HRP) solution. After 48 hours, 50 microns transverse serial sections of the brainstem were made with a vibratome. The tissue was processed with the diaminobenzidine procedure of the cobalt-glucose method. Our experiment revealed that the fibers from the basal turn terminated at the dorsomedial portion of anteroventral cochlear nuclei (AVCN), but those from the apical turn were distributed among the ventrolateral portion of the AVCN. In the posteroventral cochlear nuclei (PVCN) and dorsal cochlear nuclei (DCN), the fibers from basal to apical turns extend from the dorsal to the ventral portion of each nuclei. A distinct tonotopic arrangement could be found between the origin of cochlear fibers of each turn and their termination in the regions of the cochlear nuclei (CN). Also, the results suggested that the scratch method combined with retrograde transportation of horseradish peroxidase was useful in investigating the tonotopic arrangement of the peripheral auditory nerve in the CN.

Rapid cytochemical demonstration of cytochrome oxidase activity in pathogenic bacteria.

To develop and assess a cytochemical technique for the light microscopical detection of oxidase activity in pathogenic bacteria. Live bacterial cells were deposited on to aminopropylsilane treated glass coverslips by centrifugation, dried, then reacted with either 1% (w:v) n,n,n',n'-tetramethyl-p-phenylene diamine (TPD) or 5 mM diaminobenzidine (DAB) at 37 degrees C. The preparations were mounted in 50% glycerol and assessed by brightfield microscopy. An optimised DAB procedure (5 minutes of drying at 37 degrees C and 10 minutes of reaction time) was applied to 44 strains of organisms commonly associated with infections in man and to two fresh positive blood culture containing Gram negative bacilli. TPD gave no discernible localised reaction product and was not investigated further. With DAB, oxidase positive cells (brown) were clearly differentiated from oxidase negative cells (colourless) even in mixed preparations. The DAB technique correctly assigned 18 oxidase positive isolates (seven genera), 26 oxidase negative isolates (eight genera), and the organisms present in the two freshly positive blood cultures to their appropriate oxidase reactivity as defined by the standard macroscopic TPD technique. The cytochemical reaction seems to be a reliable indicator of the macroscopic oxidase test. It permits determination of oxidase reactivity at an early stage in the assessment of clinical material when infecting organisms can be demonstrated by microscopy. Further development of this and related cytochemical techniques could permit the provision of microbiological information that would be relevant to patient management, well in advance of conventional techniques.

Glycoconjugate histochemistry of the prostate gland in the pig

In the porcine prostate, glycoconjugates elaborated and released by the glandular cells have been studied by means of a series of histochemical methods of light microscopy. The employed methods included several routine procedures in combination with digestion by carbohydrate-degrading enzymes and peroxidase-labelled lectin diaminobenzidine procedures. In the glandular tissues, the particular glycoconjugates were shown to be secretory glycoproteins which contained vicinal diol, sulphate, and carboxyl groups and were provided with different saccharide residues such as alpha-D-mannose, alpha-D-glucose, lactose, N-acetyl-D-galactosamine, beta-D-galactose, alpha-L-fucose, N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid. These glycoconjugates were, likewise, found to exhibit positive reaction for proteins. The present histochemical results have been discussed with special reference to the histophysiological functions performed by the prostate gland in the pig.

Quantitative immunohistochemistry of metallothionein in rat placenta.

The presence of metallothionein (MT) was demonstrated in placentae from cadmium-exposed and control rats by an immunohistochemical technique, using peroxidase as label and the diaminobenzidine procedure for the staining reaction. The protein was found in different regions of the placenta, i.e. in trophoblastic labyrinth, in spongiotrophoblast and in visceral yolk sac. Cytophotometric analysis of the final reaction product revealed that the amount of MT was increased in the placental labyrinth of cadmium-exposed rats. Increases were found in both nuclei and cytoplasm of trophoblast cells in the labyrinth region. Possible roles of MT in the transport of zinc and in the carbohydrate metabolism are discussed.

Histochemistry of Glycoconjugates in the Secretory Epithelium of the Goat Bulbourethral Gland

To investigate the histochemical nature of the secretory epithelium lining the goat bulbourethral gland, glycoconjugates contained in these secretory epithelial cells have been studied by means of light- and electron-microscopic histochemistry. The methods employed involved a series of conventional staining and peroxidase-labeled lectin diaminobenzidine procedures together with combined selective methods such as digestion with enzymes. According to the results obtained in the present work, the secretory cells of the goat bulbourethral gland can be grouped into two types: cells with glycoconjugate-rich granules and those with less amounts of glycoconjugates. The gland is at least dual in the nature of its secretory activities.

The cytochemistry of glycoconjugates in the zona pellucida of murine ovarian oocytes and two-cell embryos

Changes in glycoconjugates of the zona pellucida induced by maturation, ovulation and fertilization of mouse oocytes have been studied by means of light microscopic methods of cytochemistry. These methods consisted of periodic acid-Schiff (PAS), Alcian Blue pH 1.0 and pH 2.5, and peroxidase-labelled lectin diaminobenzidine (PO-LT-DAB) procedures in combination with the digestion technique with neuraminidase. According to the results obtained, glycoconjugates of the zona pellucida of fertilized eggs contained a smaller amount of sulphate groupings than that in ovarian oocytes, whereas their reactions for sialic acid and fucose residues were significantly stronger in intensity in the former, as compared with those in the latter. The cytophysiological significance of such cytochemical changes in glycoconjugates of the zona pellucida has been discussed with special reference to its functional alterations following maturation, ovulation and fertilization.

Biochemical evidence for an endocytically inactive population of lysosomes

The peroxidase dependent, diaminobenzidine (DAB) density shift procedure was applied to the characterization of lysosomes from Chinese hamster ovary (CHO) cells. Peroxidase activity was localized in lysosomes by a 15–18 h internalization period. After treatment with DAB, the distribution of peroxidase activity in Percoll gradients was shifted, as a population, to a higher density. A bimodal distribution which included a low density population was observed for the native lysosomal enzyme β-hexosaminidase after DAB treatment. A second lysosomal enzyme, α-fucosidase, was strongly inhibited by DAB treatment with the residual activity corresponding in distribution to the light β-hexosaminidase population. The occurrence of a low density lysosomal population after the DAB procedure suggests the existence of an endocytically inactive lysosomal population in fibroblasts. Probable physiological candidates for such a population are discussed.

Horseradish peroxidase: factors affecting its distribution after retrograde infusion into the rat parotid gland.

Previous studies have shown that tight junctions of the unstimulated rat parotid gland are impermeable to retrogradely administered tracers such as myoglobin. Permeability is increased following beta-adrenergic stimulation, allowing the tracers to reach the intercellular and interstitial spaces. Reaction product of retrogradely infused horseradish peroxidase (HRP) and lactoperoxidase in found in the intercellular and interstitial spaces in both resting and stimulated glands, and many acinar and duct cells contain diffuse cytoplasmic reaction product. In this study we investigate several factors that might influence the distribution of HRP in the parotid gland. Tracer distribution was similar with HRP obtained from different suppliers, with different HRP preparations (Sigma types II, VI, VIII, and IX), and at HRP concentrations of 0.1-10 mg/ml. Inclusion of various saccharides in the infusion solution had no effect on the distribution of reaction product. Inhibition of the enzymatic activity of HRP by extraction of the heme group or reaction with hydrazine reduced but did not eliminate the extraluminal and cytoplasmic reaction product. In contrast, HRP treated with high H2O2 concentrations (0.04 M) was retained in the lumina and cytoplasmic staining was nearly abolished. Immunofluorescent localization of untreated and H2O2-treated HRP after retrograde infusion confirmed the findings obtained using diaminobenzidine procedures. These results suggest that the peroxidatic activity of HRP may damage cell membranes and tight junctions in the rat parotid gland, and indicate that permeability studies employing HRP as a tracer should be interpreted with caution.

Marginal plates in hepatic peroxisomes of Ichthyophis glutinosus (Amphibia: Gymnophiona). A cytochemical study.

The ultrastructure of hepatic peroxisomes was investigated in Ichthyophis glutinosus (Amphibia: Gymnophiona), employing perfusion fixation and the diaminobenzidine (DAB) technique for the visualization of catalase. The majority of peroxisomes is circular or rod-shaped, although elongated particles occasionally occur. They contain a finely granular matrix, lightly stained after the DAB procedure. Their mean diameter is approximately 0.25 micron. Serial sections reveal that the circular and rod-shaped peroxisomal profiles are cross and oblique sections of highly tortuous, tubular organelles exceeding 2 micron in length. In addition to tubular profiles, elongated, rectangular particles, as well as straight dumbbell-shaped organelles with distinct marginal plates are observed. They range from 900 to 1650 nm in length (mean = 1200 nm). In the flattened, thin central portion of the dumbbell-shaped particle, the peroxisomal membranes form a cisterna enclosing one or two uniformly thick marginal plates, which display a definite substructure with a periodicity of 10 nm. These findings indicate that peroxisomes in the liver of Ichthyophis exhibit a complex organization. It is suggested that the organelles undergo a specific differentiation process, morphologically characterized by the formation of enlarged segments of unusual shape.

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3'-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.

Response of chemically induced hepatocytelike cells in hamster pancreas to methyl clofenapate, a peroxisome proliferator.

Administration of N-nitrosobis (2-oxopropyl)amine during peak DNA synthesis of regenerating pancreas in hamsters has been shown to induce hepatocytelike cells in pancreas. We now present evidence to demonstrate that such cells respond to methyl clofenapate, a peroxisome proliferator. The response includes a marked proliferation of peroxisomes and enhanced activity of peroxisomal enzymes enoyl-CoA hydratase (8.5- to 13-fold), [1-14C]-palmitoyl-CoA oxidation (2.8- to 3.9-fold), catalase (1.6 to 3.4-fold), and carnitine acetyltransferase (greater than 2,000-fold). Cytochemical localization of catalase by the alkaline 3,3'-diaminobenzidine procedure and immunofluorescence localization of heat-labile enoyl-CoA hydratase showed that these peroxisome-associated enzymes are localized strictly in pancreatic hepatocytelike cells, while adjacent acinar, duct, and islet cells appeared consistently negative. Morphometric analyses of hepatocytelike cells showed a significant increase in the numerical density and an eightfold increase in the volume density of peroxisomes in methyl clofenapate treated animals. These results demonstrate that the hepatocytelike cells are responsible for the observed peroxisomal enzyme activity in pancreas of hamsters and suggest that the derepressed peroxisome specific genes in these cells respond to a peroxisome proliferator as do parenchymal cells in hamster liver.

Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: A horseradish peroxidase study

To detect the cell bodies of neurones which project to the area of the hypothalamic para-ventricular nucleus, 10–40 nl of a solution containing horseradish peroxidase and poly- l-ornithine were pressure-injected into one paraventricular nucleus of the rat. After 24 or 48 h, the enzyme remaining at the site of injection was detected by the diaminobenzidine procedure. Retrogradely transported horse-radish peroxidase was visualized by using o-dianisidine as the chromogen substrate. The extent and the intensity of labelling correlated with the apparent volume of the injection site. Labelled cell bodies were observed, ipsilateral to the injection, in the mediobasal hypothalamus, in the limbic system (lateral septum, posteromedial amygdala, ventral subiculum) and in several cell clusters in the brain stem (dorsal raphe nucleus, locus coeruleus, parabrachial nucleus, nucleus of the solitary tract and lateral reticular nucleus). In some animals, light labelling in the organum vasculosum laminae terminalis and in the subfornical organ was observed. No labelled neurones could be detected in the spinal cord.

Physiological and anatomical characteristics of primary vestibular afferent neurons in the bullfrog.

Intracellular recordings were made in the VIIIth nerve of the bullfrog (Rana catesbiana) to measure the membrane characteristics and obtain records of spontaneous and evoked spike activity of primary semicircular canal afferents. Physiological stimulation of the canals was achieved by rotating the preparation on a servomotor driven turntable with the animals' head centered in the rotational axis. The responses of each neuron to sinusoidal rotations at frequencies of 0.05Hz, 0.5Hz and for impulsive accelerations of 400 deg/sec2 were obtained. Membrane characteristics measured included the cell resting and action potential amplitude, and spike-activation threshold for applied currents. Physiologically characterized neurons were injected with horseradish peroxidase by applying pneumatic pressure and/or iontophoretic currents to the micropipettes containing 5% HRP in 1 M KCI. Following survival times of 12--48 h, the VIIIth nerve and attached vestibular end organ was removed for histochemical processing using a diaminobenzidine procedure to visualize the HRP reaction product. Light microscopy was used to discern the anatomical features of the neurons and to trace their peripheral dendritic trajectories from the ganglion to their termination(s) in the crista. Our studies have revealed that the bullfrog's primary vestibular afferents are characterized by a broad range of soma and axon diameters which correspond to an equally broad range of spontaneous and evoked activity characteristics. The largest neurons had more irregular spontaneous firing rates and consistently exhibited the greatest gain and smallest phase shifts with respect to head acceleration. These neurons consistently terminated at or near the central region of the crista. On the other hand, the smallest neurons were characterized by having the most regular spontaneous discharge patterns, the lowest gains, and greatest phase shifts with respect to head acceleration. Our findings are thus consistent with the view that the anatomical features of the primary vestibular neurons are important in determining the neuron's physiological characteristics. In terms of response dynamics our observations indicate that the receptors in the frog's crista ampullaris are heterogeneous and differentially sensitive to a wide range of stimulus frequencies.

Transganglionic and anterograde transport of horseradish peroxidase across dorsal root ganglia: A tetramethylbenzidine method for tracing central sensory connections of muscles and peripheral nerves

Horseradish peroxidase was applied either to severed sciatic nerves or to individual muscle groups in the lower leg of the rat. This horseradish peroxidase was taken up by sensory nerves and transported across the dorsal root ganglion and then anterogradely as far as central sensory terminals in the spinal cord and medulla. The dorsal root, dorsal column and the tract of Lissauer contained horseradish peroxidase labeled fibers. Following horseradish peroxidase application to the cut sciatic nerve, extraperikaryal reaction-product within the central processes of the sensory neurons was seen in the upper dorsal horn (substantia gelatinosa), lower dorsal horn, intermediate gray, ventral horn and nucleus of Clarke of the ipsilateral spinal cord; in the contralateral dorsal and ventral horns, and in the ipsilateral nucleus gracilis. Following intramuscular injections of horseradish peroxidase, the reaction-product was virtually absent in the upper dorsal horn and was concentrated in the lower dorsal horn, intermediate gray and ventral horn. These experiments show that horseradish peroxidase is transported anterogradely in significant quantities along the intact central processes of the dorsal root ganglion so that sensory terminations belonging to individual peripheral nerves or muscle groups can be traced. The demonstration of this complex transport was made possible with a sensitive histochemical method that used tetramethylbenzidine as the chromogen. Alternate sections processed with a commonly employed diaminobenzidine procedure did not show any of these central sensory connections.

Limbic and sensory connections of the inferior parietal lobule (area PG) in the rhesus monkey: A study with a new method for horseradish peroxidase histochemistry

The caudal part of the inferior parietal lobule (area PG) was injected with horseradish peroxidase (HRP) in 6 hemispheres of 5 rhesus monkeys. The retrograde transport of HRP resulted in the labeling of neurons in diverse cortical and subcortical areas. In cortex, labeled neurons were noted in prefrontal cortex (areas 8, 45, 46), in the banks of the intraparietal and superior temporal sulci, in medial parietal cortex, in cingulate cortex, in the retrosplenial area, in area TF and the caudal portions of the parahippocampal region. Subcortical sites with labeled neurons included the nucleus basalis of the substantia innominata, the claustrum, the pulvinar and intralaminar thalamic nuclei, the pretectal area, the nucleus locus coeruleus and the raphe nuclei. Although many of the labeled neurons were seen in layers IIIc and V, each cortical area had an individual laminar pattern of labeled neurons. In these experiments, a benzidine dihydrochloride (BDHC) method was used which yields a blue reaction-product at sites containing HRP. BDHC affords superior visibility of labeled neurons, and a significant improvement in sensitivity when compared to a diaminobenzidine procedure in matching series of sections. Additional sections were also stained with a method which allows the simultaneous demonstration of HRP (blue) and acetylcholinesterase (reddish-brown). These revealed that virtually all substantia innominata (nucleus basalis) neurons which project to area PG are also rich in the enzyme acetylcholinesterase. These afferents of PG may be classified into ‘sensory association’, ‘limbic’ and ‘reticular’ categories. It is argued that this arrangement of afferent input may afford a convergence of limbic and sensory information in area PG and that this may subserve a significant function in the process of sensory attention.

The histochemistry of galactose residues of complex carbohydrates as studied by peroxidase-labeled Ricinus communis agglutinin.

A peroxidase-labeled Ricinus communis agglutinin diaminobenzidine (PO-RCA-DAB) procedure has been utilized to determine the light microscopic localization of galactose residues of complex carbohydrates in a variety of tissues from different vertebrate species. In the same tissues, the localization of mannose and glucose residues was studied by means of a concanavalin A peroxidase diaminobenzidine (Con A-PO-DAB) procedure for comparison. The distribution pattern of galactose residues was found to be in distinct contrast with that of mannose and glucose residues in connective and epithelial tissues such as those involved in the umbilical cord, cartilage, aorta, comb, thyroid gland, submaxillary gland, duodenum, jejunum, colon and gill of various vertebrates. The results of the present study indicate that the PO-RCA-DAB procedure is useful in providing a means of dividing periodic acid-Schiff reactive neutral complex carbohydrates into subgroups in comparison with the intensity of their Con A-PO-DAB reaction in the histochemistry of complex carbohydrates.

Polysaccharides in lung alveoli.

Rat lung alveolar surfaces and contents were studied after using concanavalin A as a bifunctional agent to link exposed sugars to horseradish peroxidase, in accordance with a technique developed by Bernhard and Avrameas ('71). The Graham and Karnovsky ('69) diaminobenzidine procedure then was used to provide and electron-dense reaction product so as to define the distribution of complex carbohydrates in alveoli. A layer of very dense raction product was intimately associated with the outer leaflets of the luminal plasma membranes of type I and II pneumocytes. Masses of generally less dense reaction product extended irregularly into the alveolar coated by reaction product. When highly ordered tubular myelin bodies were seen, the reaction product filled all the "gutters" created by the intersentions of the membranes. Reasons are presented for believing that the intrinsic periodicity of the tubular myelin may be created and maintained by the domains ol technique. The distribution of autologous albumin, demonstrated by antibody staining by Bignon et al. ('75), apparently coincides with the carbohydrate pattern, suggesting both may be associated as a glycoprotein. The ultimate relationships between carbohydrate moieties and phospholipid membrane systems proved to be of such complexity that we believe it justifiable to think that they also may be truly complexed together.

An ultracytochemical study of the respiratory potency, integrity, and fate of the sea urchin sperm mitochondria during early embryogenesis.

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.

Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation.

Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.

A Light and Electron Microscopic Study of Peroxidase Localization in the Onion Root Tip

Peroxidase activity in the onion root tip was localized by the diaminobenzidine procedure and studied at the light and electron microscope levels. Reaction product was observed in the cell wall, on the plasmalemma, in the Golgi apparatus cisternae and vesicles, in young and developing vacuoles, in the endoplasmic reticulum, and on both soluble and membrane-bound ribosomes. The reaction product at the various cellular and subcellular sites occurs in distinct tissue and developmental patterns.