bluuebunny/crossref_metadata_2025 huggingface dataset:
Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations.
Authors: Llewellyn-Smith I J , Costa M , Furness J B
Published: April 2011

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.
A fluorescence staining method for the demonstration and measurement of lysosomal enzyme activities in single cells.
Authors: Luyten G P , Hoogeveen A T , Galjaard H
Published: April 2011

A cytochemical fluorescence method is described that makes possible simple, rapid, and specific demonstration and measurement of the activities of a wide variety of lysosomal enzymes in single cells using 4-methylumbelliferyl derivatives as substrates. The validity of the method and a number of applications using normal and mutant human cells are presented.
Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors.
Authors: Papermaster D S , Schneider B G , DeFoe D , Besharse J C
Published: April 2011

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.
Models for the self-assembly of basement membrane.
Authors: Yurchenco P D , Tsilibary E C , Charonis A S , Furthmayr H
Published: April 2011

Basement membranes contain a number of intrinsic macromolecular components which are unique to these structures and which cooperatively assemble into specific heteropolymeric matrices. Type IV collagen triple helical monomers bind together at their amino-terminal, carboxy-terminal, and lateral domains to form a lattice-like array. Laminin, in a two-step process, binds to itself at its terminal globular domains to form polymers and also binds collagen at two distinct sites along the collagen chain. Heparan sulfate proteoglycan has been found to bind both collagen and laminin, suggesting a reversible crosslinking function. On the basis of the data derived from self-association studies, it is possible to begin considering models for the assembly and structure of these ubiquitous matrices.
Amphibian limb regeneration curves generated by the scanning laser acoustic microscope.
Authors: Slobin J A , Stocum D L , O'Brien W D
Published: April 2011

A scanning laser acoustic microscope was used to generate growth curves for amphibian limb regeneration. The mechanical properties of the specimen were converted into a visual image that was suitable for the measurements of limb growth. Bone, cartilage, muscle, dermis, and epidermis can be visualized.
Compartmentation and characterization of different proteoglycans in bovine arterial wall.
Authors: Völker W , Schmidt A , Buddecke E
Published: April 2011

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.
Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts.
Authors: Castagnino C G , Berria M I
Published: April 2011

A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.
Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre)osteoclast identification.
Authors: van de Wijngaert F P , Burger E H
Published: April 2011

Fixed, undecalcified mouse long bones were embedded in glycol methacrylate (GMA), sectioned, and incubated for acid phosphatase in the presence or absence of tartrate, to investigate the feasibility of tartrate-resistant acid phosphatase as a histochemical marker for osteoclast identification. Naphthol AS-BI phosphate was used as the substrate and hexazonium pararosanaline as coupler. Cytocentrifuge preparations of mouse, rat, and quail bone marrow or frozen and GMA sections of mouse splenic tissue were used as controls to specify acid phosphatase activity. After adequate fixation, acid phosphatase activity sensitive to tartrate inhibition (TS-AP) was demonstrated in macrophages from spleen, bone marrow, and loose connective tissue surrounding bone rudiments. Acid phosphatase activity resistant to tartrate inhibition (TR-AP), was detected in multi-nuclear osteoclasts and in some mononuclear cells from bone marrow and periosteum. In cytocentrifuge preparations and frozen sections of mouse spleen, TR-AP was demonstrated after simultaneous incubation with substrate and tartrate. In GMA sections, however, TR-AP could only be demonstrated after pre-incubation with tartrate before application of substrate. We suggest that histochemical demonstration of TR-AP versus TS-AP on GMA-embedded bone sections by means of a pre-incubation method can be used as an identification marker of (pre)osteoclasts. Plastic embedding is recommended for its excellent preservation of morphology and enzyme activity.
Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence.
Authors: Coulter H D , Elde R P
Published: April 2011

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.
Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine.
Authors: Becker J , Schuppan D , Benzian H , Bals T , Hahn E G , Cantaluppi C , Reichart P
Published: April 2011

The aim of the present study was to characterize the composition of the organic matrix in alveolar jaw bone and dentine using antibodies against pro-collagens Types I and III and collagens Types IV, V, and VI. After demineralization of oral hard tissues in 0.2 N HCl, antigenicity was well preserved and the distribution of the pro-collagens and collagens could be demonstrated. Staining for pro-collagen Type I was prominent around osteoblasts and in pre-dentine, indicating active de novo synthesis of Type I pro-collagen. Pro-collagen Type I was ubiquitous but was less abundant in bone and dentine, whereas pro-collagen Type III was seen only in areas of bone remodeling, in peritubular spaces, and in pre-dentine. Type IV collagen was limited to the basement membranes of vessels in osteons and bone marrow. Type V collagen was detected neither in pre-dentine nor in bone. In contrast, Type VI collagen was found in dentine and bone, showing a faint but homogeneous staining which, similarly to pro-collagen Type III, was pronounced around osteoblasts and in pre-dentine, areas of active bone and dentine formation. This study showed that the organic matrix of dentine and bone contains Type VI as well as Type I collagen. Pro-collagen Type III (and to a lesser extent collagen Type VI) is transiently produced during new formation and remodeling of oral hard tissues, and disappears once the matrix calcifies. Type I pro-collagen qualifies as a general marker protein for increased osteoblastic activity. We conclude that immunostaining for the different collagen/pro-collagen types can be used to assess normal or abnormal stages of bone/dentine formation.
Gold labeling of microtubules in cleaved whole mounts of cortical root cells.
Authors: Traas J A , Kengen H M
Published: April 2011

A method is described for localizing microtubules using gold-labeled antibodies in combination with anti-tubulin. Cortex cells of Equisetum hyemale are broken open while still in buffer, after initially being attached to poly-L-lysine-coated grids. Thus, the cytoplasm becomes accessible to the antibodies. After application of the antibodies, the cleaved cells are post-fixed, stained, dehydrated, and critical point-dried. Different fixation procedures are compared: fixation in paraformaldehyde, in glutaraldehyde, and in glutaraldehyde followed by a sodium borohydride reduction step. All three methods result in good labeling of the microtubules, with low backgrounds. However, organization of the cytoplasm is best preserved in cells fixed in glutaraldehyde without sodium borohydride treatment. The method is highly suitable for studying the membrane-bound cytoskeleton because detergent extraction and/or embedding are avoided.
Combined technique of immunohistochemistry and autoradiography for the simultaneous detection of steroid hormone receptors and their ligand.
Authors: Gasc J M , Ennis B W , Baulieu E E , Stumpf W E
Published: April 2011

A combined technique of immunohistochemistry and autoradiography was applied to detect progesterone target cells in tissue sections. The radioactively labeled progestin [3H]-Organon 2058 and the progesterone receptor, revealed by antibodies to the receptor molecule, were localized simultaneously in identical cells on the same tissue section. Technical details that make possible combined detection of the nuclear antigen, present in very small amounts, simultaneously with its steroid ligand are described.
Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper.
Authors: Clark R K , Tani Y , Damjanov I
Published: April 2011

Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength of the buffer used could alter this binding. Furthermore, as powdered non-fat milk has been proposed as a possible blocking agent for nonspecific binding of ABC, we sought to determine if it would adequately block that binding in our system. The initial experiments showed that nonspecific binding of ABC to proteins transferred to nitrocellulose membranes was due to the avidin component of the ABC; little, if any, binding was seen if biotin alone was incubated with these blots. The spurious binding was shown to be primarily due to the high affinity of avidin to proteins electroblotted to nitrocellulose, when incubated in low-salt buffers. Low-fat milk added to the buffer reduced overall nonspecific reactivity but produced additional artifacts in the form of bands that were not seen in other preparations. Nonspecific avidin binding to proteins transferred to nitrocellulose can therefore be effectively reduced by adding extra salt to buffers, whereas the addition of non-fat dry milk does not seem suitable for this purpose.
Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments.
Authors: Hare J D
Published: April 2011

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.
Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes.
Authors: Gallyas F , Wolff J R
Published: April 2011

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.
Ultrastructural variations of the urothelial membrane in essential fatty acid (EFA)-deficient rats. A digital densitometric study.
Authors: Kalinec F , Calderón J H , Monis B
Published: April 2011

The present report deals with a densitometric study of the ultrastructural images of the urothelial membrane of rats in the following experimental conditions: (1) EFA-deficient (EFAD) rats; (2) EFA-sufficient (EFAS) rats; and (3) EFAD rats that were fed the EFAD diet for 30 weeks and received an EFAS diet for the following 10 weeks (EFAD/S group). On electron micrographs of the transitional epithelium of ureters and urinary bladder of these rats, optical density (OD) profiles of the urothelial unit membrane were recorded and digitized using a computer-controlled microdensitometer with a solid-state self-scanned photodiode array sensor. A Gaussian curve was adopted as a model for the distribution of electron-dense material in each osmiophilic leaflet. Gaussian parameters were used to estimate the thickness of the urothelial membrane and of each osmiophilic leaflet, and the amount of electron-dense material and the maximal electron density present in each leaflet. In EFAS rats, the thick urothelial membrane was asymmetric like that of the normal, resulting from a greater thickness of the outer leaflet and a greater electron density of the cytoplasmic one. In EFAD rats, a loss of the characteristic ultrastructural asymmetry and a decrease of the total thickness of the unit membrane were detected. These changes were partially reversed in the EFAD/S rats.
Thymic hormone-containing cells. VIII. Effects of colchicine, cytochalasin B, and monensin on secretion of thymulin by cultured human thymic epithelial cells.
Authors: Savino W , Dardenne M
Published: April 2011

The intracellular pathway for secretion of thymulin, a thymic hormone, was studied in primary cultures of human thymic epithelial cells by experimentally blocking the movement of secretory vesicles within these cells. These cultures were subjected to cytoskeleton inhibitors, such as colchicine and/or cytochalasin B, that block the directed movement of secretory vesicles, or to monensin, an ionophore that specifically perturbs the traffic of Golgi-derived vesicles. Both cytoskeleton inhibitors partially prevented thymulin secretion into the culture supernatants, and their effects were dose-dependent. Moreover, the percentage of thymulin-containing cells (evaluated by immunofluorescence with a zinc-specific anti-thymulin monoclonal antibody), as well as the fluorescence intensity within these cells, was significantly higher than observed in control cultures, suggesting that the hormone was accumulated in the cytoplasm, thus facilitating its detection. Similar results were obtained with monensin. These results, together with the recent identification of high molecular weight proteins reacting with anti-thymulin antibodies, suggest that thymulin is secreted via the following intracellular pathway: a precursor is synthesized at the level of the granular endoplasmic reticulum; it migrates to the Golgi complex, from which it is released within hormone-containing vesicles; the vesicles incorporate zinc, move towards the cell membrane, and eventually fuse with it. This sequence of events characterizes the classical phenomenon of exocytosis.
A quantitative immuno-electronmicroscopic study of amylase and chymotrypsinogen in peri- and tele-insular cells of the rat exocrine pancreas.
Authors: Posthuma G , Slot J W , Geuze H J
Published: April 2011

Malaisse-Lagae demonstrated in 1975 that peri-insular (PI) cells and tele-insular (TI) cells produce amylase (Am) and chymotrypsinogen (Ch) in a different ratio. These biochemical measurements are in contradiction with recent observations of Bendayan (1985), who found that the Am/Ch ratio measured with the protein A-gold technique applied to ultrathin Epon sections was the same in PI and TI cells. We have previously shown (Posthuma et al., 1984) that experimentally induced changes in Am and Ch content of rat pancreas are quantitatively reflected by immuno-gold labeling of zymogen granules in cryosections. Here we applied the same technique to compare the Am/Ch labeling density ratios in PI and TI pancreatic cells. To ascertain constancy of experimental conditions, we used ultrathin cryosections from tissue blocks consisting of TI and PI tissue elements. Consecutive sections of these blocks were alternatively immunolabeled for Am and Ch, using protein A-gold as marker. The density of gold particles over zymogen granules of both PI and TI cells was measured. It appeared that the Am/Ch labeling density ratio was significantly lower in PI than in TI cells. This difference resulted from a lower Am labeling as well as higher Ch labeling density over zymogen granules in PI cells.
Assessment of cell cycle-associated antigen expression using multiparameter flow cytometry and antibody-acridine orange sequential staining.
Authors: Bauer K D , Clevenger C V , Williams T J , Epstein A L
Published: April 2011

A novel approach which enables direct assessment of the differential expression of cellular antigens in noncycling (G0) and cycling cell subpopulations is presented. The method involves flow cytometric analysis and sorting of cells stained by use of indirect immunofluorescence, followed by restaining using acid acridine orange, to relate the immunofluorescence of sorted lymphoid subpopulation(s) to cell proliferation status (i.e., G0 vs. G1 vs. S vs. G2 and M). In the present study, this technique successfully identifies the proliferation-associated modulation of a heterochromatin-associated antigen in pokeweed mitogen-stimulated human lymphoid cultures. The potential utility of this method for documenting early antigenic changes associated with the G0-G1 transition is discussed.
Immunological characteristics and histological distribution of human hair fibrous proteins studied with anti-hair keratin monoclonal antibodies HKN-2, HKN-4, and HKN-6.
Authors: Ito M , Tazawa T , Ito K , Shimizu N , Katsuumi K , Sato Y
Published: April 2011

By use of a mouse hybridoma technique, monoclonal antibodies were produced against hair fibrous proteins, which were extracted from normal human hairs. Three monoclonal antibodies, designated as HKN-2, HKN-4, and HKN-6, were chosen and used to investigate the immunological characteristics of hair fibrous proteins. Epidermal fibrous proteins were also extracted from human sole callus horny materials. Hair or epidermal fibrous proteins were electrophoretically separated on polyacrylamide gels with sodium dodecyl sulfate. By immunoblot analyses, HKN-2 and HKN-4 marked the electrophoretic bands of both proteins; however, HKN-6 reacted only with the bands of hair fibrous proteins. Immunohistochemically, all three monoclonal antibodies stained the keratogenous zone of anagen hairs. HKN-6 did not react with any other skin components or with tissues of other organs examined. Although HKN-2 showed reactions with skin epithelial tissues, except for epidermal basal cells and secretory cells of sweat glands, the reactivity of HKN-2 was limited within the skin. HKN-4 displayed a broad crossreactivity with all of the skin epithelial cells and various epithelial cells of other organs. These findings indicate that some components of hair fibrous proteins are immunologically specific to hair cells, whereas others broadly crossreacted with the fibrous proteins of other skin epithelial cells or with those of various epithelial cells. The anti-hair keratin monoclonal antibodies seem useful to examine the differentiation patterns of epithelial cells and tissues.
Quantitative histochemistry of benzaldehyde dehydrogenase in hepatocellular carcinomas of vinyl chloride-treated rats.
Authors: Chieco P , Normanni P , Moslen M T , Maltoni C
Published: April 2011

Hepatocarcinogenesis in rats treated with several chemicals is associated with changes in aldehyde dehydrogenase (AlDH) activity, particularly heterogeneous expression of a "tumor specific" phenotype that is very active with aromatic aldehydes, e.g., benzaldehyde (Bz). Objectives of this study were first, to determine if liver cancers in vinyl chloride-treated rats also expressed this AlDH phenotype, and second, to quantitate the NAD- and NADP-dependent AlDH activity for the substrates Bz and acetaldehyde (Ac) in the cancers and surrounding tissue. Small cubes of tissue containing well-differentiated hepatocellular carcinoma were obtained from five Sprague-Dawley rats exposed to 2500 ppm vinyl chloride for 55 weeks. An optimized procedure was developed for AlDH histochemistry. Frozen sections were preincubated in nitroblue tetrazolium/acetone and then incubated at 20 degrees C in viscous polyvinyl alcohol media containing buffer, phenazine methosulfate, sodium azide, substrate, coenzyme, and nitroblue tetrazolium. Background activity was evaluated by omission of substrate. Activity was quantitated by computer-assisted microscopic photometry. All five carcinomas had heterogeneous staining of NADP- and NAD-dependent BzDH and AcDH activity, with clusters of very high-activity cells. The magnitude of staining in the high-activity neoplastic cells was at least tenfold greater for BzDH-NADP and about twofold greater for BzDH-NAD, AcDH-NADP, and AcDH-NAD than the staining in other liver cells. More neoplastic cells had high BzDH than high AcDH activity. Only BzDH-NADP was localized predominantly to the carcinoma.
Varicella-zoster viral glycoprotein envelopment: ultrastructural cytochemical localization.
Authors: Montalvo E A , Parmley R T , Grose C
Published: April 2011

The periodate-thiocarbohydrazide silver proteinate (PA-TCH-SP) method was used to study the envelopment process in varicella-zoster virus-infected human melanoma cells. Viral envelopment could be seen at two sites, the nuclear membrane and at virus-induced intracytoplasmic vacuoles. Virus-associated glycoconjugates were detected by the PA-TCH-SP method at the plasmalemma and on the inner membrane of the intracytoplasmic vacuoles. Virion envelopes acquired at the nuclear membrane were PA-TCH-SP negative, whereas those acquired at intracytoplasmic vacuoles were PA-TCH-SP positive. All virions found inside these vacuoles contained periodate-reactive envelopes. Release of virions into the extracellular space, where virtually all virions were PA-TCH-SP positive, appeared to be via exocytosis. Thus, the PA-TCH-SP method identifies glycoprotein incorporation at specific cytoplasmic vacuoles distinct from nuclear envelope, endoplasmic reticulum, and Golgi lamellae. These results suggest that envelopment within the cytoplasm is a stage in the assembly of the varicella-zoster virion.
Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L.
Authors: Pesacreta T C , Bennett A B , Lucas W J
Published: April 2011

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.
Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons.
Authors: Knyihár-Csillik E , Bezzegh A , Böti S , Csillik B
Published: April 2011

Thiamine monophosphatase (TMPase) has been selectively localized in small dorsal root ganglion cells and in their central and peripheral terminals. Light microscopic localization of TMPase, and its alterations due to transganglionic effects, are identical with those of fluoride-resistant acid phosphatase (FRAP), but are not contaminated by the ubiquitous lysosomal reaction inevitable in trivial acid phosphatase-stained sections. TMPase is inhibited by 0.1 mM NaF, which is slightly less than the concentration needed to inhibit FRAP (0.2-0.4 mM). It is assumed that TMPase and FRAP are identical enzymes. In the perikaryon of small dorsal root ganglion cells, TMPase is located in the cisterns of the endoplasmic reticulum and in the Golgi apparatus. The central terminals of these cells are scalloped (sinusoid) axon terminals, surrounded by membrane-bound TMPase activity. Central terminals outline substantia gelatinosa Rolandi throughout the spinal cord, as well as the analogous nucleus spinalis trigemini in the medulla. TMPase-active central terminals outline "faisceau de la corne postérieure" in the sacral cord, as well as Lissauer's tract in the thoracic, upper lumbar, and sacral segments, and the paratrigeminal nucleus and the terminal (sensory) nucleus of the ala cinerea in the brainstem. Peripheral terminals displaying TMPase activity are fine nerve plexuses of C fibers. The TMPase activity of the central terminals disappears after dorsal rhizotomy in the course of Wallerian degeneration, and is depleted in the course of transganglionic degenerative atrophy (after transection of the related peripheral sensory nerve). TMPase is an outstanding genuine marker for the study of transganglionic regulation in Muridae.
Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist.
Authors: Niendorf A , Dietel M , Arps H , Lloyd J , Childs G V
Published: April 2011

Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.
Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures.
Authors: Gown A M , Garcia R , Ferguson M , Yamanaka E , Tippens D
Published: April 2011

We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.
Characterization of retrograde axonal transport of antibodies in central and peripheral neurons.
Authors: Wenthold R J , Skaggs K K , Reale R R
Published: April 2011

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.
Ultrafast microwave energy fixation for electron microscopy.
Authors: Login G R , Stavinoha W B , Dvorak A M
Published: April 2011

We demonstrate that microwave (MW) energy can be used in conjunction with chemical cross-linking agents to fix tissue blocks rapidly for electron microscopy in as brief a time as 26 msec. The optimal ultrafast MW fixation methodology involved immersing tissue blocks up to 2 mm3 in dilute aldehyde fixative and immediately irradiating the specimens in a 7.3 kW MW oven for 26-90 msec, reaching a fixation temperature range of 32-42 degrees C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hr at 25 degrees C. Potential applications for this new fixation technology include investigation of rapid intracellular processes (e.g., vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (e.g., antigens and enzymes).
Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro.
Authors: Kersken H , Vilmart-Seuwen J , Momayezi M , Plattner H
Published: April 2011

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.
Localization of L-arginine-glycine amidinotransferase protein in rat tissues by immunofluorescence microscopy.
Authors: McGuire D M , Gross M D , Elde R P , van Pilsum J F
Published: April 2011

Creatine is a major component of energy metabolism and enzymes involved in its synthesis have therefore been of considerable interest. L-arginine-glycine amidinotransferase, commonly called transamidinase, catalyzes the first reaction in the biosynthesis of creatine. This first reaction is believed to occur in the kidney because of the high concentration of transamidinase in that tissue. Transamidinase activity is also found in many other tissues of the rat, but its role in these tissues is not known. Immunochemical studies with antisera and monoclonal antibodies were used to confirm and refine our understanding of the presence of transamidinase in rat tissues. Immunofluorescence histochemistry was performed to localize transamidinase immunoreactivity within specific tissues including cells in the proximal tubules of the kidney, hepatocytes of the liver, and alpha cells of the pancreatic islet. Immunochemical studies with monoclonal antibodies confirm localization of transamidinase immunoreactivity in the proximal tubules of the kidney. The localization of such immunoreactivity in specialized cells yields insight into possible physiological role(s) of transamidinase in the rat.
Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo.
Authors: Kersken H , Momayezi M , Braun C , Plattner H
Published: April 2011

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.
Detection of insulin synthesis in mammalian anterior pituitary cells by immunohistochemistry and demonstration of insulin-related transcripts by in situ RNA-DNA hybridization.
Authors: Budd G C , Pansky B , Cordell B
Published: April 2011

Insulin or highly homologous transcripts is shown to be synthesized in cultures of mammalian anterior pituitary cells using cloned insulin-specific cDNA probes and nucleic acid cytochemistry. The insulin-hybridizing cells are less abundant than the growth hormone-producing cells, occurring in the cultures at approximately one tenth the frequency. Immunocytochemistry demonstrates that insulin or insulin-like proteins is also synthesized by the cultured pituitary cells and that the insulin immunoreactivity is contained within secretory granules. It appears that many of these secretory granules are concentrated around the periphery of the cell, unlike the insulin-containing granules in pancreatic B-cells.
Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives.
Authors: Clark R K , Damjanov I
Published: April 2011

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.