bluuebunny/crossref_metadata_2025 huggingface dataset:
STAINING PROPERTIES OF BRAIN TISSUE BEFORE AND AFTER TREATMENT WITH POTASSIUM DICHROMATE
Authors: LANDSMEER J. M. F. , GIEL R.
Published: April 2011

This paper deals with the influence of the pH and of salts on the staining of brain tissue with basic and acid stains, before and after treatment with potassium dichromate. The pH value and the ratio UO2++/Ba++ for destaining identify the ionized groups of tissue. In frozen sections of the brain the metachromatic staining of myelin sheaths results from interaction of phosphate containing compounds with toluidin blue. In the nerve cells also, phosphate groups in the Nissl substance and the nucleus form the substrate of staining, though it is probable that carboxyl groups are also involved in the process. In paraffin sections, after treatment with alcohol, the phosphate-containing substances of the myelin sheaths are dissolved and a residual protein skeleton remains, which contains carboxyl groups. Frozen sections stained with acid stains at a pH lower than 4.5 present an over-all staining of all elements except for the myelin sheaths which remain "empty". In paraffin sections the residual protein skeleton is also stained by acid stains. Brain tissue fixed in dichromate before contact with alcohol shows a staining picture with acid stains identical with that of frozen sections, which means a selective staining of axons, cells and gray substance. The precipitated myelin seems to enclose the protein skeleton and to prevent an acid stain from gaining the positive groups. While myelin regularly shows phosphate groups, precipitated myelin contains carboxyl groups. The results of the findings are discussed as to their significance in general and also in relation to Alzheimer-Mann-Häggqvist's method for staining brain tissue.
STUDIES ON ARTHOROPOD CUTICLE. XII. ASH ANALYSES AND MICROINCINERATION
Authors: RICHARDS A. GLENN
Published: April 2011

1. The ash amounts to only 0.365% of the dry weight of cleaned, water-washed cuticles of Sarcophaga (fly) larvae, to only 0.095% of cuticles of Limulus (Horseshoe crab). These values are considerably lower than ash values for whole insects or for cellular structures in general.

2. Elemental analyses show at least 25 elements in the ash of these cuticles. Ten are present in amounts > 1% of the ash. Magnesium is the predominant cation.

3. The percentage values for the elements do not agree well with the per cent composition of the solids of sea water or arthropod blood or calcified cuticles.

4. The cations are closely balanced by the anions. This implies that the ash components are largely present as salts which are either insoluble or sorbed.

5. Chitin carefully purified and washed in polyethylene vessels is ash-free.

6. The cuticulin sublayer of the epicuticle (thin surface layer, Fig. 3) leaves a relatively large amount of ash which is qualitatively different from that of the thick underlying procuticle. The iron of the cuticle is concentrated here.

7. The procuticle (Fig. 3) leaves relatively little ash. This ash residue shows a laminar organization similar to that of normal cuticle.

8. After sclerotization (hardening and darkening) the ash residue of the epicuticle seems unchanged but that of the procuticle is greatly reduced except in the tonofibrillae (muscle attachment points). Sclerotization, then, is accompanied not only by partial dehydration as reported by previous workers but also by extrusion of most of the ash components from the exocuticle.
STRUCTURAL AND CHEMICAL STUDIES ON THE CONNECTIVE TISSUE OF MARINE SPONGES
Authors: GROSS JEROME , SOKAL ZELDA , ROUGVIE MALCOLM
Published: April 2011

1. The mesoglea of the marine sponge, S. graminea, resembles in its organization and composition the connective tissue of vertebrates in that it is composed of collagen fibers and fibrils embedded in an amorphous matrix containing carbohydrates.

2. Two morphologically distinct forms of spongin fibers, designated spongin "A" and spongin "B", were demonstrated to be members of the collagen class, structurally by x-ray diffraction and electron microscopy, and chemically by their hydroxyproline and glycine content as well as by the general amino acid pattern. Ratios of glycine to hydroxyproline were 1.6 and 1.8 for spongin "A" and "B" respectively. Spongin "A" is a long unbranched fibril of uniform width, of the order of 200 A revealing an axial period of 625 A by small-angle x-ray diffraction and of about 650 A by electron microscopy. Spongin "B" is a large branched fiber 10-50 µ in width composed primarily of bundles of thin unbranched filaments less than 100 A wide. "B" fiber fragments occasionally showed an axial period in the collagen range although x-ray preparations did not yield a low-angle pattern.

3. Content of hexosamine, hexose, pentose and uronic acid was determined in both fiber types and in the amorphous matrix. Glucosamine, galactosamine, glucose, galactose, mannose, fucose, arabinose and uronic acid were identified chromatographically in both spongin "A" and in the amorphous substance. A very small amount of amino sugar plus glucose and galactose were identified in spongin "B". All sugars with the exception of arabinose are found in mammalian skin connective tissue.

4. Spongins "A", "B" and the amorphous matrix were readily separated by tryptic digestion and water extraction.
CHROMOSOMIN AND THE FEULGEN REACTION
Authors: KASTEN FREDERICK H.
Published: April 2011

Evidence is presented, using the acidic dye acid fuchsin, that the Feulgen reaction is not based on acid-base mechanisms as suggested by a few authors. Various extraction, enzymatic and other procedures are used to show that the Feulgen reaction is specific for deoxyribonucleic acid when using either acid fuchsin or basic fuchsin in Schiff's reagent. There is no evidence here that any other nuclear component is stained in the Feulgen reaction.
STUDIES IN HISTOCHEMISTRY: XLII FURTHER STUDIES ON THE DETERMINATION OF SUCCINIC DEHYDROGENASE IN MICROGRAM AMOUNTS OF TISSUE AND DISTRIBUTION OF THE ACTIVITY IN THE BOVINE ADRENAL
Authors: GLICK DAVID , NAYYAR SOM N.
Published: April 2011

Optimal conditions for the determination of succinic dehydrogenase activity in microgram quantities of intact tissue by the method of Defendi and Pearson were studied, and as a consequence certain modifications were found to be advantageous. The improved procedure was compared with that of Jardetzky and Glick which was designed for use on comparable quantities of disrupted tissue. Both procedures gave results in relative agreement with one another, but the former was somewhat simpler, technically, and more sensitive.

The methods were applied to the quantitative histological distribution of the enzyme in the adrenal gland of the steer, and a maximum concentration was observed in the fasciculata with a symmetrical falling off through the zones on both sides to minima in the medulla and capsule. Thus a species difference was noted since earlier work on the rat adrenal showed a maximum in the reticulanis.
HISTOCHEMICAL METHODS FOR ACID PHOSPHATASE
Authors: GOMORI GEORGE
Published: April 2011

Results obtained by the PbS method and by the post-coupling method of Rutenburg and Seligman are closely similar. However, there are minor differences which appear to be due partly to the presence of more than one acid phosphatase in the tissues, partly to diffusion artifacts. Results of techniques recommended so far for the histochemical demonstration of acid phosphatase must be interpreted with caution, especially with respect to finer details.
THE MOBILIZATION AND TURNOVER TIMES OF CELL POPULATIONS IN BLOOD AND BLOOD-FORMING TISSUE
Authors: YOFFEY J. M.
Published: April 2011

1. A description is given of a technique for performing absolute and differential counts of the nucleated cells of guinea-pig bone marrow. Emphasis is placed on the importance of using animals of uniform age in marrow studies.

2. In a 400 gm. male guinea-pig of the Dunklin-Hartley strain, the total marrow volume is 7.0 cc., the total blood volume 28.8 cc., and exchanges of cell population between marrow and blood can be readily calculated on the basis of these figures.

3. Thoracic duct lymphocyte production per day is about 4 times the total lymphocytes normally present in the blood and about one sixth to one eighth of the total marrow lymphocyte population.

4. Lymphocytes in bone marrow are practically all small, and are not formed in the marrow, but come to it from the blood stream.

5. In respect to granulocytes, the marrow possesses about 150 times the number normally present in the blood, and two thirds of this myeloid reserve consists of mature or almost mature cells which can be rapidly discharged into the blood stream.

6. Leucocytosis Promoting Factor (Menkin) evokes in 4 hours a dual response by the marrow, namely a discharge of granulocytes into the blood and an uptake of lymphocytes from the blood. But most of the granulocytes thus entering the blood rapidly leave it.

7. Unlike the granulocytes, there is no large erythroid reserve in the bone marrow, and the quantitative data indicate that under normal conditions erythrocyte production just about keeps pace with requirements.

8. In animals subjected to hypoxia at the Jungfraujoch, about 3 days elapse before there is an appreciable discharge of additional erythrocytes into the blood. The nucleated erythroid cells of the marrow begin to increase on the second day, and reach a peak on the fourth day.

9. Morphological evidence indicates the existence of "Transitional" lymphocytes, intermediate between the small lymphocyte and the blast cell.
CELL TURNOVER IN EPITHELIAL POPULATIONS
Authors: HOOPER C. E. STEVENS
Published: April 2011

In its simplest terms, cell turnover in a tissue is the process whereby cell formation is balanced by cell loss. Close examination of the turnover systems in epithelia, such as the epidermis and intestinal epithelium, reveals that the mechanism of the process is, in fact, very complex and may differ widely between epithelia of different morphology and function. The existence of cell turnover in an epithe1ium may be shown morphologically. Cell formation is indicated by the presence of mitotic activity. This may be restricted to one region of the epithelium, as in the deeper layers of the epidermis and the crypts of the intestinal epithelium. Regions of cell loss are indicated in the epidermis by cornification and death of the surface cells, and in the intestinal epithelium, by the presence of extrusion zones at the tips of the villi.

Cell turnover in an epithelium is probably an inherent property rather than a repair process induced by trauma. However, the rate of cell turnover may be altered by a number of internal influences (e.g., hormones) as well as by factors in the external environment (e.g., temperature, feeding). Alteration of the turnover rate may be shown by changes in mitotic activity, or in size of the cell population of the epithelium. Thus, in contrast with non-renewing tissues, whose architecture is always constant, the histologic picture of an epithelium undergoing renewal may vary with the interplay of factors controlling its cell turnover.
CELLULAR LOCALIZATION OF β-GLUCURONIDASE IN RAT TISSUES
Authors: FISHMAN WILLIAM H. , BAKER JOHN R.
Published: April 2011

1. A detailed study has been made of the use of the substrate, 8-hydroxyquinoline glucosiduronic acid as a means of localizing the enzyme, β-glucuronidase in cells and tissues of the rat.

2. Conditions for preparing the substrate solution have been developed which yield a stable mixture capable of giving satisfactory service for a period of months.

3. The introduction of chloral hydrate into the formalin fixative solution contributes greatly to the preservation of the enzyme and the successful demonstration of its cellular sites.

4. Certain refinements in technique, e.g., removal of precipitate artifact, prerefrigeration in substrate solution have greatly improved the convenience, reproducibility and uniformity of the cytochemical result.

5. Success in dealing with friable tissues in these histochemical studies has been achieved by employing a gelatin-embedding technique, sections being cut from the frozen block.

6. A systematic study of over 30 tissues and organs of the adult rat reveals striking localizations in specialized cells and tissues, e.g., Purkinje cells, pancreatic islet cells, uterine endometrium, as well as more uniform intense staining in organs such as liver and renal cortex. In most instances, the results agree with the findings of Seligman who employed a different technique.

7. The finished preparations do not deteriorate and are suitable for the observation of fine histological detail.
Peripheral distribution of dermatan sulfate proteoglycans (decorin) in amyloid-containing plaques and their presence in neurofibrillary tangles of Alzheimer's disease.
Authors: Snow A D , Mar H , Nochlin D , Kresse H , Wight T N
Published: April 2011

We used a polyclonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the small dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristic lesions in Alzheimer's brain. All antibodies demonstrated positive decorin immunostaining in both the amyloid deposits of neuritic plaques (NPs) and the filamentous structures within neurofibrillary tangles (NFTs). Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout NPs containing amyloid fibrils, decorin was primarily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorin was also immunolocalized to the paired helical and straight filaments within NFTs and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque.
Alternative staining methods for Lowicryl sections.
Authors: Horowitz R A , Woodcock C L
Published: April 2011

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.
Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.
Authors: Lumpert C J , Glas-Albrecht R , Eisenmann E , Plattner H
Published: April 2011

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.
Biological significance of dermal Merkel cells in development of cutaneous nerves in human fetal skin.
Authors: Narisawa Y , Hashimoto K , Nihei Y , Pietruk T
Published: April 2011

We detected epidermal Merkel cells in 12-week fetuses with monoclonal antibodies (MAb) against simple epithelium keratin and epithelial membrane antigen. In 15-week fetuses these Merkel cells began to descend into the dermis and expressed nerve growth factor receptors (NGF-R). At approximately the same time, cutaneous nerves, as detected with an MAb against neurofilaments, extended from the subcutaneous trunk and branched to form the subepidermal nerve plexus. The expression of NGF-R on dermal Merkel cells preceded their connection with immunoreactive small nerves. Initially, most of these fine nerve endings were directed towards dermal Merkel cells. In 23-week fetuses the subepidermal nerve plexus was well developed and immunoreactive dermal Merkel cells began to disappear. At all stage of fetal development the epidermal Merkel cells did not strongly express NGF-R. We postulate that dermal Merkel cells play an inductive and a promotional role in development of the cutaneous nerve plexus in the upper dermis.
Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.
Authors: Visa N , Fibla J , Santa-Cruz M C , Gonzàlez-Duarte R
Published: April 2011

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.
Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.
Authors: Marshak D W
Published: April 2011

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.
Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.
Authors: Schmidt H H , Gagne G D , Nakane M , Pollock J S , Miller M F , Murad F
Published: April 2011

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.
Cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human iris and ciliary body.
Authors: Kivelä T , Fuchs U , Tarkkanen A
Published: April 2011

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.
The ontogeny and distribution of surfactant protein B in human fetuses and newborns.
Authors: Stahlman M T , Gray M E , Whitsett J A
Published: April 2011

The distribution of immunoreactive surfactant-associated protein B (IR-SP-B) was studied immunohistochemically in 120 subjects from 10 weeks of gestation to 7 postnatal months with a polyclonal antibody against human SP-B. Electron microscopy (EM) was done in 72 subjects to document the presence of Type II cells containing lamellar bodies. Fetuses of less than 18 weeks' gestation showed no immunostaining. Beginning at 18 weeks, non-mucous cells of tracheal glands immunostained in a few instances. Fetuses of 19 through 23 weeks showed progressive immunostaining of cells lining terminal airways. Infants 26-40 weeks who died with or without pulmonary pathology showed immunostaining of Type II cells and bronchioloalveolar (BA) portal cells of the respiratory bronchioles. In infants with hyaline membrane disease (HMD) who died less than 12 days after birth, occasional tracheal gland cells, BA portal cells, and mature and relining Type II cells immunostained. In bronchopulmonary dysplasia (BPD), BA portal cells, relining Type II cells, macrophages, and luminal material immunostained. Occasional tracheal and bronchial gland cells and Clara cells immunostained. The appearance of IR-SP-B at mid-gestation correlated with differentiation of Type II cells. There was good correlation of immunostaining with the presence of lamellar bodies on EM. Accelerated maturation of the lung was often associated with premature rupture of membranes (PROM).
Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.
Authors: Haller E M , Shelley S A , Montgomery M R , Balis J U
Published: April 2011

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.
Endocytosis and intracellular transport of transferrin across the lactating rabbit mammary epithelial cell.
Authors: Seddiki T , Delpal S , Ollivier-Bousquet M
Published: April 2011

To study the transcytosis and segregation of ligand in the mammary epithelial cell, endocytosis and intracellular transit of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried across the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles.
Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung.
Authors: Voorhout W F , Veenendaal T , Kuroki Y , Ogasawara Y , van Golde L M , Geuze H J
Published: April 2011

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.
Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.
Authors: Quatacker J R , Annaert W G , Miserez B J , De Potter W P
Published: April 2011

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.
Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.
Authors: Knowles G C , McCulloch C A
Published: April 2011

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.
Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.
Authors: Asari A , Miyauchi S , Miyazaki K , Hamai A , Horie K , Takahashi T , Sekiguchi T , Machida A , Kohno K , Uchiyama Y
Published: April 2011

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.
Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.
Authors: Winston D C , Proctor G B , Garrett J R , Schulte B A , Thomopoulos G N
Published: April 2011

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.
Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.
Authors: Yamamoto K , Völkl A , Fahimi H D
Published: April 2011

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.
A new fluorescence method for alkaline phosphatase histochemistry.
Authors: Murray G I , Ewen S W
Published: April 2011

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.
The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.
Authors: Schellens J P , Frederiks W M , Van Noorden C J , Vreeling-Sindelárová H , Marx F , McMillan P J
Published: April 2011

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.
GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.
Authors: Laurie S M , Mixon M B , Castle J D
Published: April 2011

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.
Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.
Authors: Vandré D D , Burry R W
Published: April 2011

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.
Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.
Authors: Burry R W , Vandré D D , Hayes D M
Published: April 2011

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.
Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.
Authors: Iida H , Hatae T , Shibata Y
Published: April 2011

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.