bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.
Authors: Tranzer J P , Richards J G
Published: April 2011

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.
Arterial hypotension induced by horseradish peroxidase in various rat strains.
Authors: Deimann W , Taugner R , Fahimi H D
Published: April 2011

The influence of various commercial preparations of horseradish peroxidase upon the arterial pressure has been investigated using direct intra-arterial pressure recordings in Nembutal anesthetized rats of different strains. A marked hypotension was noted in Wistar and Sprague-Dawley rats when sufficiently high doses (5-10 mg/100 g body weight) were injected intravenously. This effect was observed with both highly purified (Sigma type VI, Boehringer Reinheitsgrad I) as well as less pure preparations (Sigma type II, Boehringer Reinheitsgrad II). Prior injection of promethazin, an antihistamine, prevented the drop in arterial pressure. Wistar/Furth rats appeared almost resistant to the hypotensive effect of Sigma preparations but developed moderate to marked hypotension with both types of Boehringer horseradish peroxidase. When instead of Nembutal, ether anesthesia was used only a transient mild hypotension developed and the animals recovered within 7-10 min. No effect was noted in Syrian hamsters receiving injections with the same doses of horseradish peroxidase. Electrophoresis revealed marked variations in the isozyme composition of different horseradish peroxidase preparations. Since the exact nature of the hypotensive agent remains unknown, it is suggested that the arterial pressure be monitored before extensive systematic application of this tracer in vascular permeability studies in rats.
Peroxisome development in the regenerating pars recta (P3 segment) of proximal tubules of the rat kidney.
Authors: Reddy J K , Rao M S , Moody D E , Qureshi S A
Published: April 2011

The development of peroxisomes, lysosomes and endocytic vacuoles in regenerating cells of the pars recta (P3 segment) of proximal tubules, in rats given a single interperitoneal injection of d-serine (80 mg/100 g.b.wt), was studied by light and electron microscopy using cytochemical methods. Rapid proliferation of cells occurred between 2 and 5 days after d-serine induced tubular necrosis; by day 6 almost all injured tubules were re-epithelialized with flat or low cuboidal cells. Peroxisomes and lysosomes were not observed during the period of rapid cell multiplication i.e., between 2 and 6 days after d-serine injection. Restitution of mitochondrial population preceded the development of peroxisomes in the newly regenerated cells of P3 tubules. Maximum development of peroxisomes occurred between 9 and 14 days after d-serine injection. The formation of peroxisomes appeared to correlate closely with the differentiation of apical endocytic vacuoles and the brush border. Lysosomes in the regenerated cells of P3 tubules were the last to develop.
Acrolein as a fixative for enzyme cytochemistry.
Authors: Saito T , Keino H
Published: April 2011

Since acrolein can penetrate more quickly and deeply into tissue blocks than glutaraldehyde, the possibility of the use of this aldehyde as a prefixative in enzyme cytochemistry was reinvestigated. At low concentrations, acrolein preserves the activities of the enzymes investigated, including those of glucose-6-phosphatase, which is known as one of the most vulnerable to aldehyde fixation; thus, acrolein is usable in enzyme ultracytochemistry. Enzyme activities are also preserved in tissues fixed with acrolein and glutaraldehyde combined. The rapid penetration of acrolein enables fixation in larger tissue blocks and provides greater freedom in specimen selection, especially important advantages when encountering heterogeneous materials as in pathology.
Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. I. Aspects on the distribution of dopamine and noradrenaline nerve terminals.
Authors: Löfström A , Jonsson G , Fuxe K
Published: April 2011

Using the fluorescence histochemical technique of Falck and Hillarp, a quantitative microfluorimetric study of the catecholamine fluorescence in the median eminence has been performed. On the basis of morphologic criteria, the median eminence was subdivided into various areas from which the microfluorimetric measurements were made; the subependymal layer, the medial and lateral palisade zone of the rostral and the central and caudal region of the median eminence (for definitions of the various areas and regions, see Anatomical Subdivision). The highest fluorescence intensities were recorded from the lateral palisade zone, indicating that this area has the most dense catecholamine innervation, whereas the lowest fluorescence intensities were recorded from the subependymal layer. Dopamine-beta-hydroxylase inhibition produced by FLA-63, fusaric acid or diethyldithiocarbamate resulted in all cases in a 50-70% reduction of the catecholamine fluorescence in the subependymal layer, whereas only minute effects were observed in the lateral palisade zone. In the medial palisade zone, these treatments generally led to a substantial reduction (30-50%) of the catecholamine fluorescence. Basal hypothalamic deafferentation according to Halasz, or lesioning of the ventral catecholamine bundle, produced an almost complete disappearance of the fluorescence in the subependymal layer, while both procedures were largely ineffective in affecting the catecholamine fluorescence in the lateral palisade zone. On basal hypothalamic deafferentation the catecholamine fluorescence in the medial palisade zone was markedly reduced (40-60%), while the ventral bundle lesions were less efficient in this respect. From the present results it is suggested that the subependymal layer is mainly innervated by noradrenaline nerve terminals and the lateral palisade zone is mainly innervated by dopamine nerve terminals, whereas the medial palisade zone receives a mixed innervation of dopamine and noradrenaline terminals, the dopamine proportion being in the order of 50-75% of total catecholamine content.
Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states.
Authors: Löfström A , Jonsson G , Wiesel F A , Fuxe K
Published: April 2011

Catecholamine nerve terminals in the rat median eminence have been studied using the fluorescence histochemical technique of Falck and Hillarp in combination with quantitative microfluorimetry. The catecholamine fluorescence intensities recorded from various parts of the median eminence were all found to be within the linear part of the dopamine or noradrenaline concentration-fluorescence relationship as studied in an agar-albumin model system. The catecholamine fluorescence was also found to disappear with time in an exponential manner following tyrosine hydroxylase inhibition produced by alpha-methyl-p-tyrosine methylester (H44/68). Similar results were obtained when measuring the dopamine decline by mass fragmentography in the median eminence after H44/68 treatment. These results and analysis of fluorescence frequency histograms strongly indicate that the catecholamine fluorescence values recorded are proportional to the catecholamine concentration. It is concluded that the microfluorimetric technique used is a reliable method for catecholamine quantitation in discrete nerve terminal areas of the median eminence. The main advantages of the technique are that a high sensitivity and quantitative data on the transmitter content can be obtained in strict relation to the neuroanatomy. Measurement of the catecholamine fluorescence disappearance after H44/68 was used to evaluate catecholamine turnover during various endocrine states. The results showed that two dopamine systems with different transmitter turnover may be distinguished. Tuberinfundibular dopamine neurons projecting to the lateral palisade zone were thus shown to have a slower turnover than those projecting medially to the capillary loops. No definite changes in catecholamine turnover were observed after adrenalectomy and castration in the male, although there was a tendency toward increased noradrenaline turnover in both states. During pregnancy an increase in noradrenaline as well as dopamine turnover was noted. The present results therefore give further evidence for the view that catecholamine nerve terminals in the median eminence may participate in the regulation of gonadotrophin secretion.
Dimethyl sulfoxide-lead tetraacetate method for histochemical oxidation of polysaccharides.
Authors: Malinin G I
Published: April 2011

Oxidation of fixed tissues and unfixed peripheral blood smears by 1% (w/v) lead tetraacetate in dimethyl sulfoxide for 10 to 60 min resulted in facile induction of tissue carbonyls readily demonstrable with Schiff's reagent and by sodium bisulfite addition reaction, followed by toluidine blue staining at controlled pH. Tissue carbonyls represented a broad range of oxidation-labile substrates and included neutral polysaccharides (glycogen). The oxidation procedures were not destructive to tissues and were characterized by technical simplicity and consistent reproducibility, thus affording a substantial improvement over the hitherto used methods of histochemical oxidation by lead tetraacetate.
Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.
Authors: Anastasia-Sawicki J , MacIntyre R J
Published: April 2011

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.
The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.
Authors: Shimada M , Kihara T , Watanabe M , Kurimoto K
Published: April 2011

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.
Tannic acid effect on membrane of cell surface origin in guinea pig megakaryocytes and platelets.
Authors: Fedorko M E , Levine R F
Published: April 2011

Plasma membranes in isolated guinea pig megakaryocytes and washed platelets are poorly stained with the usual methods used to outline cell membranes. The addition of tannic acid and calcium to the initial fixative is useful to enhance electron density of all surface-derived membrane systems in these cells. The method described here shows that the increased electron denisty of membrane after fixation in the presence of tannic acid occurs both at the cell surface and along the invaginated membrane systems.
The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein.
Authors: Whitaker J N
Published: April 2011

The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.
Histochemical characteristics of the striated inclusions of adrenoleukodystrophy.
Authors: Johnson A B , Schaumburg H H , Powers J M
Published: April 2011

The straited accumulations in adrenal cortical cells and brain macrophages that are characteristic of adrenoleukodystrophy have been studied histochemically in cryostat sections to seek leads for the biochemical identification of the striated material. It stained pale pink with oil red O and did not stain with the Schultz cholesterol procedure or periodic acid-Schiff technique. By utilizing the birefringence of the accumulations as a marker, it was determined that, unlike natural cholesterol and cholesterol esters, the striated material was resistant to acetone and ethanol extraction. It was readily soluble, however, in nonpolar solvents such as n-hexane and chloroform. These findings indicated that the material was most probably a lipid, and they suggested that sequential extraction of adrenoleukodystrophy adrenal and brain with acetone and then n-hexane could be used to isolate this material in relatively pure form. Based on this lead, biochemical studies have just revealed a fatty acid abnormality in adrenoleukodystrophy which appears to be unique to this genetic disease.
Approach to three-dimensional mapping of quantitative histochemical measurements applied to studies of the cochlear nucleus.
Authors: Godfrey D A , Matschinsky F M
Published: April 2011

After reviewing the historical development of some procedures for localizing quantitative histochemical measurements, an approach is presented which combines some aspects of these procedures to make possible a permanent, objective record of the location of each sample. The method further allows direct comparisons to be made with drawings of other nearby sections during the process of sampling. Illustrations are given of various applications to a study of the cochlear nucleus. A particular benefit of the approach is to facilitate comparison of chemical with anatomical and physiologic data.
Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver.
Authors: Roels F
Published: April 2011

In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.
Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.
Authors: Silverman A J
Published: April 2011

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.
Immunocytochemistry of the pituitary glycoprotein hormones.
Authors: Moriarty G C
Published: April 2011

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.
Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy.
Authors: Pickel V M , Joh T H , Reis D J
Published: April 2011

The monoamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and tryptophan hydroxylase (TrH) were immunocytochemical localized in dopaminergic, noradrenergic and serotonergic neurons of rat brain by light and electron microscopy. In dopaminergic and serotonergic neurons, the respective synthesizing enzymes. TH and TrH, were distributed throughout the cytoplasm of the neuronal perikarya, dendrites, axons and terminals. The most selective accumulation of reaction product for the specific enzyme was associated: (a) in perikarya with endoplasmic reticulum, Golgi apparatus and microtubules, (b) in processes with microtubules, and (c) in terminals with dense granules or clear vesicles. The labeled terminals were characterized by their content of labeled organelles and the absence of synaptic junctions. In noradrenergic neurons, both TH and DBH were localized in the perikarya, similar to TH in dopamine neurons. TH and DBH differed in their localization within proximal axons and dendrites in that TH was associated with microtubules but DBH was not. These results provide ultrastructural evidence to suggest that monoamines may be: (a) synthesized by enzymes which are associated with different organelles depending on the portion of the neuron and the type of enzyme; (b) synthesized in both axons and dendrites and (c) released from terminals without postsynaptic membrane specializations.
Sequential protein analysis from single identified neurons of Aplysia californica. A microelectrophoretic technique involving polyacrylamide gradient gels and isoelectric focusing.
Authors: Rüchel R
Published: April 2011

Electrophoresis in polyacrylamide gradient gels and isoelectric focusing in polyacrylamide gels of capillary size are powerful tools for the analysis of molecular weight and charge properties of small protein samples. This is demonstrated using identified neurons from the abdominal ganglion of the sea hare Aplysia californica. Certain cell-specific peptides, which are considered to be neurosecretory, have been shown to be water soluble when ethylene glycol was employed as a mobilizing agent. Although the mode of action of ethylene glycol is not yet understood, this method may be of value for various extraction procedures. The application of a new staining method that is preferential for separations of sodium dodecyl sulfate-proteins yields information about the charge of water-insoluble proteins which has so far been inaccessible. Preliminary results gained by a small, two-dimensional mapping procedure as well as optical density separation patterns of two different nuclear protein fractionation from a single isolated nucleus outline further possibilties of the microgel techniques.
Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.
Authors: Bayer E A , Skutelsky E , Wynne D , Wilchek M
Published: April 2011

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.
Single-strand nuclease action on heat-denatured spermiogenic chromatin.
Authors: Hunter J D , Bodner A J , Hatch F T , Balhorn R L , Mazrimas J A , McQueen A P , Gledhill B L
Published: April 2011

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.
Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.
Authors: McKeever P E , Garvin A J , Spicer S S
Published: April 2011

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.
Ultrastructural localization of catalase and L-alpha-hydroxy acid oxidase in microperoxisomes of Hydra.
Authors: Hand A R
Published: April 2011

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.
Tissue fixation with diimidoesters as an alternative to aldehydes. II. Cytochemical and biochemical studies of rat liver fixed with dimethylsuberimidate.
Authors: Hand A R , Hassell J R
Published: April 2011

Rat liver fixed with dimethylsuberimidate (DMS) was studied to investigate the use of diimidoesters as dixatives for light and electron microscopic cytochemistry. Paraffin sections of liver fixed with DMS at pH 9.5 were weakly stained with the ninhydrin-Schiff procedure, indicating extensive reaction of NH3+ groups with the fixative. Nuclei were strongly strained by the Feulgen procedure, with no background [corrected] reaction. In contrast, glutaraldehyde fixation resulted in a significant background reaction in the cytoplasm and nuclei in controls for the Schiff-based stains. DMS-fixed liver stained intensely for glycogen with the Periodic acid-Schiff procedure, and biochemical analysis of glycogen retention and extractability indicated that DMS retained considerably more glycogen in sections than glutaraldehyde. DMS-fixed liver incubated for thiamine pyrophosphatase activity revealed reaction product in ER cisternae, Goli saccules and bile canaliculi. Peroxisomes were strongly reactive for catalase activity after incubation in diaminobenzidine medium, and reaction product of glucose-6-phosphatase activity was considerably greater following DMS fixation than after glutaraldehyde. Biochemical studies revealed up to twice as musch residual activity of glucose-6-phosphatase after DMS fixation. These results suggest that DMS may be useful as a primary fixative for certain cytochemical procedures.
Comparative immunoenzymatic localization of prolactin and growth hormone in human and rat pituitaries.
Authors: Martin-Comin J , Robyn C
Published: April 2011

Twelve human and twelve rat pituitaries were stained by an immunohistochemical method using a rabbit anti-ovine prolactin serum, a rabbit anti-human growth hormone serum and a sheep anti-rabbit immunoglobulin serum conjugated with horseradish peroxidase. On the same pituitary section, growth hormone cells were stained brown by using 3-3'-diaminobenzidine as peroxidase substrate, and prolactin cells were stained purplish blue by using 4-chloro-1-naphtol. Growth hormone cells outnumbered prolactin cells, especially in human pituitaries where the proportion is at least 10:1. No cells containing both brown granules stained for growth hormone and blue granules stained for prolactin were found in any of the sections examined. In the fetal pituitaries, there was no apparent hypertrophy of the prolactin cells, although the circulating levels of the hromone are known to be as high in the fetus at term as in the mother and much higher than in nonpregnant women.
Cytochemical reactions of human hematopoietic cells in liquid culture.
Authors: Willcox M B , Golde D W , Cline M J
Published: April 2011

Growth of human bone marrow in liquid suspension cultures has been used to study normal hematopoietic cell differentiation and abnormalities in blood diseases. A variety of cytochemical stains were applied to human marrow cells cultured in vitro for up to 14 days. AS-D- CHLOROACETATE ESTERASE AND ALPHA-NAPHYHYL BUTYrate esterase were most useful in distinguishing different cell lines in culture. Peroxidase activity disappeared with mononuclear phagocyte morphogenesis and diminished with culture in intermediate and mature granulocytes. Acid phosphatase activity and methyl greed pyronin staining intensity increased with macrophage maturation.
A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts.
Authors: Granström G , Linde A
Published: April 2011

In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.
Ultrastructural localization of acetylcholinesterase in cultured cells. I. Embryo muscle.
Authors: Sawyer H R , Golder T K , Nieberg P S , Wilson B W
Published: April 2011

Several techniques were employed to examine the localization of acetylcholinesterase (EC 3.1.1.7, AChE) in cultured chick embryonic skeletal muscle. Glutaraldehyde produced the best cellular preservation but less enzyme activity was lost when the cells were fixed in paraformaldehyde. Two staining methods were examined: in one (Karnovsky MJ, Roots L: J Histochem Cytochem 12:219, 1964) potassium ferricyanide was added with the primary reactants, and in the other (Tsuji S: Histochemistry 42:99, 1974) the potassium ferricyanide was added at the end of the staining procedure. Localizations of AChE were similar with both stains; activity was present in the nuclear envelope, the perinuclear sarcoplasm, the sarcoplasmic reticulum, subsurface vesicles and bound outside the cells. /owever, a granular artifact was found with the method of Karnovsky and Roots that did not appear with the method of Tsuji. The localization of AChE are consistent with kinetic data that AchE binds, moves and is released from cultured muscle fibers.
Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction.
Authors: Enerbäck L , Gustafsson B , Mellblom L
Published: April 2011

A simple technique for the condensation of cellular 5-hydroxytryptamine (5-HT) with formaldehyde gas is described. The technique, which is especially suited for quantitative cytofluormetric studies, involves the generation of formaldehyde gas from dry paraformaldehyde in a closed reaction vessel with the addition of a measured quantity of water. The fluorescence yield of 5-HT was tested at various humidities. Optimal results were obtained with the addition of 100 mg water to a 1000 ml reaction vessel containing 6 g of dry paraformaldehyde. A major advantage of the method if the fact that the humidity during the reaction can be precisely controlled. The fluorescence yield of 5-HT, tested over a 50 day period showed excellent reproducibility. The stoichiometry of the reaction was tested by comparison of cytofluormetic data with that obtained by analysing the 5-HT content of pooled mast cells with an independent biochemical method. A highly satisfactory correlation (r = 0.96) was obtained within the range of 0.1 to 4 pg of 5-HT per cell. The limit of sensitivity of the cytofluorometric method was found to be of the order of 10(-13) g, and was determined by the fluorescence blank of the mast cells. This contributes to between 10 and 30 per cent of the total fluorescence emission from mast cells containing about 0.2 pg of 5-HT.
Optical properties of normal and injured cells. Application of cytographic analysis to cell viability and volume studies.
Authors: Penttila A , McDowell E M , Trump B J
Published: April 2011

A cell spectrophotometer (Cytograf Model 6300 A, Bio/Physics Systems, Inc.) was tested in a cytotoxic assay using Ehrlich ascites tumor cells as a model system. Several cellular conditions associated with volume expansion, staining of cellular components and fixation of cells were applied and the magnitude of the scattering and extinction signals were tested in these diverse cellular conditions. The magnitude of the scattering pulse of a cell spectrophotometer was found to be greatly dependent on the staining color and intensity of cellular components with vital dyes or following osmium tetroxide fixation. When the absorption wavelength of the vital dye was close to the wavelength used in the cell spectrophotometer (about 630 nm), dead stained and living nonstained cell populations were completely separated from each other. The magnitude of the extinction pulse was greatly dependent on the state (normal, injured cells) and staining intensity (vital dye staining, osmium fixation) of cellular components. The magnitude of the extinction pulse was reduced from that in normal cells when cells were treated with p-chloromercuribenzene sulfonic acid or in a hypotonic solution that caused a marked volume expansion of injured cells. When cells were fixed with a mixture containing glutaraldehyde and osmium tetroxide the cellular components of normal and injured cells turned black and in these conditions the magnitude of the extinction signal was in a linear correlation to the cross-sectional area of cells. In the present study, the cell spectrophotometer proved to be an efficient method for estimation of cellular viability, based on different scattering properties of cells, offering the advantages of high speed and precision. Demonstration of the use of a variety of vital dyes having diverse extinction properties with capabilities to differentiate between living and dead cells has indicated the potential use of the cell spectrophotometer in cytotoxic assay. Modification of the magnitude of the extinction signal in the cell spectrophotometer also shows great promise for accurate automated size determination of both normal and injured cells. Previously, determination of the size of injured cells has been beset with methodologic errors.
Immunoelectronmicroscopic localization of alpha-actinin in skeletal muscle cells.
Authors: Lane B P , Elias J , Drummond E
Published: April 2011

The ultrastructural distribution of alpha-actinin in skeletal myocytes was studied by a sensitive method that permits localization of antigenic proteins in intact fixed cells rather than in glycerol estracted tissues or isolated cell fractions. Binding to the Z line of specific antiserum produced in rabbits immunized with highly purified alpha-actinin was demonstrated by deposition at this site of a reaction product of horseradish peroxidase that had been coupled to antigen-binding fragments of sheep anti-rabbit gamma globulin.
Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.
Authors: Coulson P B , Bishop A O , Lenarduzzi R
Published: April 2011

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.
Cytochemical and biochemical latency of alkaline phosphatase in granules of neutrophilic leucocytes.
Authors: De Jong A S , Van der Ploeg M
Published: April 2011

Biochemical and cytochemical latency of alkaline phosphatase activity was demonstrated in periotoneal guinea pig leucocytes. By incorporation of a cell homogenate containing intact granules or a cell sonicate into polyacrylamide films, cytochemical latency up to 75% could be demonstrated.
Quantitative histochemical studies of the hypothalamus enzymes of acetylcholine metabolism.
Authors: Packman P M , Godfrey D A , Williams A D , Matschinsky F M
Published: April 2011

The quantitative histochemical distribution of acetylcholinesterase and choline acetyltransferase activity has been measured in individual hypothalamic nuclei and median eminence, as well as in entire hypothalamic sections by a mapping technique. There was an 18-fold range of nuclear choline acetyltransferase activity with highest activities in the lateral preoptic nucleus and median eminence. There was a nine-fold range of nuclear acetylcholinesterase activity with highest activities in the lateral preoptic and magnocellular nuclei and lowest activity in the median eminence. The substantial gradients of choline acetyltransferase activity found in the hypothalamus indicate the importance of using a technique that provides an objective, permanent record of contiguous sample locations thereby allowing detailed analysis of tissue areas using, but not dependent on, anatomical boundaries.
Demonstration of two 3,3'-diaminobenzidine oxidation reactions associated with photosynthetic membranes in anaerobic light-grown Rhodospirillum rubrum.
Authors: Stevens F J , Pankratz H S , Uffen R L
Published: April 2011

Photosynthetic membranes of anaerobic light-grown Rhodospirillum rubrum oxidized 3,3'-diaminobenzidine. When glutaraldehyde-treated cells were exposed to 3,3'-diaminobenzidine in the light aerobically, the oxidation appeared to occur by two systems. One reaction was stimulated by white light and the second required molecular oxygen. The O2-dependent activity was inhibited by KCN.
Nucleoside diphosphatase in the onion root tip. II. The effect of inhibitors on isoenzymes.
Authors: Troyer G D , Goff C W , Klohs W D
Published: April 2011

An electrophoretic study was performed to determine the number of isoenzymes of nucleoside diphosphatase in onion root extract. Five bands exhibiting nucleoside diphosphatase activity were detected when gels were incubated with inosine diphosphate, uridine diphosphate, guanosine diphosphate or cytidine diphosphate as substrates. These consisted of a single fast migrating band (band one), a group of three intermediate migrating bands (bands two, three and four) and a single slow migrating band (band five). Gels incubated with adenosine diphosphate, thiamine pyrophosphate, inosine monophosphate, guanosine monophosphate and cytidine monophosphate showed only two bands (bands one and five). Inhibitor studies showed that sodium fluoride inhibited bands one and five but not bands two, three and four. Conversely, 1% (v/v) glutaraldehyde inhibited bands two, three and four but did not inhibit bands one and five. These results suggest that two separate groups of onion nucleoside diphosphatase isoenzymes occur which have different substrate specificities and are selected against by certain inhibitors.
Uranaffin reaction: a new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines.
Authors: Richards J G , Da Prada M
Published: April 2011

Amine storage organelles of aldehyde-fixed rabbit platelets have a strong affinity for uranyl ions before their dehydration and appear highly electron-dense when examined by electron microscopy; both the matrix and membrane of these organelles are intensely stained. This affinity, which is also shown by platelets of other species, including healthy reserpinized platelets, which contain no cytochemically demonstrable amine, show a positive uranaffin reaction. However, platelets and megakaryocytes of strains with storage pool deficiency (low adenosine-5'-triphosphate), including patients with Hermansky-Pudlak syndrome, are uranaffin negative. The cytochemical reaction, probably the result of an interaction between UO2++ ions and phosphate groups of 5'-phosphonucleotides, is also observed in adrenal medulla, sympathetic nerve terminals and ganglion cells, suggesting that the technique will be of considerable value in identification of aminergic neurons and in further elucidation of amine storage mechanisms.
Separation and partial characterization of fractions derived from frog lung homogenates. A possible marker system for amphibian pulmonary surfactant.
Authors: Hitchcock K R , Parsons W J
Published: April 2011

The purpose of this study is to determine if inframammalian vertebrate (amphibian) lung contains certain nonspecific esterases that have been identified as enzyme markers for mammalian (rat and mouse) pulmonary surfactant. Density gradient centrifugation procedures were utilized to concentrate any surface-active material in frog lung homogenates. Lipid and protein analyses of one of the derived fractions and of pulmonary lavage fluid were consistent with other techniques indicating that these preparations were surface active. A comparison of the nonspecific esterases in the derived fractions and the pulmonary lavage fluid allowed the identification of a nonspecific esterase that has an electrophoretic mobility comparable to one of the nonspecific esterases already identified as an enzyme marker for mammalian (rat and mouse) pulmonary surfactant. These results indicate that these enzyme markers may be useful in the further investigation of the surfactant systems of other inframammalian vertebrates.
Immunocytochemistry of the olfactory marker protein.
Authors: Monti-Graziadei G A , Margolis F L , Harding J W , Graziadei P P
Published: April 2011

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.