bluuebunny/crossref_metadata_2025 huggingface dataset:
Block staining with p-phenylenediamine for light microscope autoradiography.
Authors: Dilley R , McGeachie J
Published: April 2011

A study was designed to test the suitability of p-phenylenediamine (pPd) as a block stain for light microscope autoradiography. This was done to obviate the conventional method of staining through the emulsion with histological stains after exposure and development. Ten rats were injected with 3H-thymidine and 1 to 3 days later were perfused with glutaraldehyde. Tissue samples were processed by three different schedules: 1) direct embedding in Araldite-Epon; 2) postfixation in OsO4 before embedding; 3) postfixation in OsO4 and staining in 1% pPd before embedding. Autoradiographs of these tissues showed that pPd-treated tissues were well stained with clearly defined cellular detail. The nuclear region was pale-staining, which highlighted the overlying silver grains. There was no evidence of chemography and the nuclear labeling intensity did not differ significantly from the fixed and post-fixed tissues. It was concluded the pPd is a most useful block prestain to use for light microscope autoradiography.
The mystery of the unstained Golgi complex cisternae.
Authors: Locke M , Huie P
Published: April 2011

The Champy-Maillet OsKI reaction has been used upon Golgi complexes to show two kinds of staining. It stains material being processed as it passes along the secretory pathway of the rough endoplasmic reticulum (RER) and Golgi cisternae (GC) up to crystallization in secretory vesicles. It also stains separately the environment within parts of the GC. This GC staining may occur in all compartments (transition vesicles, saccules, condensing vacuoles), but it is characteristically missing from any one of them. The unstained cisternae may be explained if outer saccules are made from either stained or unstained transition vesicles, both of which occur. The presence of empty, unstained transition vesicles is dictated by the surface to volume ratios of microvesicles in relation to saccules. Most transition vesicles must return their membrane to the endoplasmic reticulum, but from time to time it is presumed that they fuse to make a saccule. Saccules, stained and unstained, then mature through the stack. OsKI reactions with tissues and test molecules suggest that in the RER and GC the stain detects labile--S . S--bridges before they lock the tertiary configuration of proteins.
Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.
Authors: Roth J
Published: April 2011

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.
Immunoelectron microscopic exploration of the Golgi complex.
Authors: Slot J W , Geuze H J
Published: April 2011

Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.
The ontogeny of regulatory peptide-containing cells in the human fetal stomach: an immunocytochemical study.
Authors: Stein B A , Buchan A M , Morris J , Polak J M
Published: April 2011

The antral and fundic regions of the stomachs from 24 human fetuses were examined by immunocytochemistry for the presence of three regulatory peptides (gastrin, somatostatin, and glucagon) and one amine (serotonin (5-HT)) in the epithelial endocrine cells. Gastrin- and somatostatin-containing cells were present at the earliest stage examined (8 weeks). Gastrin cells were restricted to the antrum, while somatostatin cells were found in both the antrum and the fundus. Glucagon-immunoreactive cells were detected from 10 weeks and were confined to the fundus. Serotonin-containing cells were found in both the antrum and the fundus from 11 weeks. Changes in the number of immunoreactive gastrin and somatostatin cells during gestation were quantified. The increase in the number of cells/mm length of vertically sectioned mucosal epithelium best reflects the change in cell population. The peptides and amine studied were found to be contained in separate cell types. Electron microscopic examination of the peptide-containing cells showed that the fetal cells contain granules of similar morphology to their adult counterparts.
The neuronal endoplasmic reticulum: its cytochemistry and contribution to the endomembrane system. I. Cell bodies and dendrites.
Authors: Broadwell R D , Cataldo A M
Published: April 2011

The endoplasmic reticulum (ER) and its contribution to the endomembrane system (i.e., membranes of cell organelles) in the neuron have been investigated in brains of mice by applying electron microscopic enzyme cytochemistry for demonstration of glucose-6-phosphatase (G6Pase) activity. The phosphohydrolytic activity of G6Pase is a well-known cytochemical marker for the ER in numerous cell types. Of the different substrates employed, glucose-6-phosphate and mannose-6-phosphate were the only two with which G6Pase reaction product was seen in the neuronal ER and organelles related morphologically to the ER. G6Pase activity in cell bodies and dendrites was localized consistently within the lumen of the nuclear envelope, rough and smooth ER, lamellar bodies, hypolemmal and subsurface cisternae, and frequently in the cis saccules of the Golgi apparatus. The G6Pase reactive ER appeared as a network of saccules and tubules pervading the cell body and its dendrites. Possible membrane continuities were identified between the ER and the other reactive structures, including the cis half of the Golgi apparatus. Neither G6Pase activity nor reactive ER was associated with the trans Golgi saccules or GERL. G6Pase activity thus serves as a reliable marker for the perikaryal and dendritic ER and related structures. These observations support the theory that the ER is an integral component of the neuronal endomembrane system associated with the transfer of membrane or membrane molecules among intracellular compartments, the packaging and transport of exportable protein, and energy metabolism. G6Pase activity in the ER of axons and terminals is considered in detail in part two of this study.
Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.
Authors: Bré M H , Pouphile M , Delpech S , Lefort-Tran M
Published: April 2011

In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin.
Identification of macrophages in sections of rabbit lung using acetoacetylated lipoproteins.
Authors: Hass A J , Davis H R , Elner V M , Glagov S
Published: April 2011

Macrophages were labeled in sections of rabbit lung with acetoacetylated low density lipoprotein (LDL), a marker internalized by cultured macrophages but not by other connective tissue cells. Using a modified technique, thin slices of fresh rabbit lung were incubated in 3,3'-dioctadecylindocarbocyanine (DiI)-labeled, acetoacetylated LDL, fixed in paraformaldehyde, and sectioned. Alveolar macrophages incorporated the fluorescently labeled, modified LDL, but surrounding stroma and parenchyma did not stain. Our results indicate that DiI-labeled, acetoacetylated LDL may be used to identify mononuclear phagocytes in tissue sections.
Osteogenic periosteum esterase activity: a comparative morphological and cytochemical study of bone cells in situ on rat proximal tibiae and in smears.
Authors: Ries W L
Published: April 2011

Mononucleate phagocytes are presently thought to participate, along with osteoclasts, in bone resorption. Similar cells may also be precursors to osteoclasts. Esterase is a hydrolytic enzyme commonly found in most phagocytes and cells such as these have been shown to degrade bone matrix in vitro. Therefore, periosteal cells of remodeling proximal tibiae in rats were examined for esterase activity. Stain reactions were compared both in situ and in smears. Percentages of different cell types were obtained by counting cells in Wright-Giemsa-stained smears. Osteoclasts and mononucleate phagocyte-like cells made up approximately 20% of the isolated cells. Cells resembling preosteoblasts, mononucleate phagocytes, and osteoblasts stained for esterase activity with light to moderate intensity. Osteoclasts and mononucleate osteoclast-like cells were strongly esterase positive. The esterase was characterized as being type B by using nonorganophosphorus inhibitors of the various esterase types. The addition of esterase to the list of enzyme activities of cells in close proximity to osteoclasts lends further support to the hypothesis that mononucleate cells also participate in bone resorption.
A new methodological approach for studying axonal transport: cytofluorometric scanning of nerves.
Authors: Larsson P A , Goldstein M , Dahlström A
Published: April 2011

A new technique for studying axonal transport has been developed. The technique, which is based on histofluorescence techniques, enables the measurement of several different accumulated substances and parameters within a single nerve in relation to a nerve crush or local cooling. Any substance that can be made to fluoresce can be measured. The tissue is treated according to the formaldehyde-induced fluorescence method of Hillarp and Falck for visualization of monoamines, or according to the indirect immunofluorescence method. For immunofluorescence the nerve is cryostat-sectioned and various sections can be incubated with primary antisera against different antigens. After incubation and mounting the sections are placed in a cytofluorimeter (Leitz MPV II). They are passed under a measuring slit at a steady speed by a motor driven cross-table. The fluorescence intensity passing through the measuring slit is continuously registered by a recording unit with an integrator. This recorder produces a graphical nerve accumulation profile, and the area under the profile, relating to the fluorescence, is expressed in arbitrary units. This article presents data on the accumulation of noradrenaline, dopamine beta-hydroxylase, and tyrosine hydroxylase in crush-operated rat sciatic nerve. The time-course accumulations for noradrenaline (visualized by the Falck and Hillarp method) and dopamine beta-hydroxylase (visualized by immunofluorescence) demonstrated a striking similarity, which is to be expected since the two substances are stored in the same organelle. Tyrosine hydroxylase (visualized by immunofluorescence) showed a slower accumulation with time, but faster than would be expected had the enzyme been 100% soluble. Colchicine but not lumi-colchicine blocked the transport of noradrenaline organelles. With the new scanning technique we have the potential to study accumulation profiles of several different substances within a single nerve. Morphometric data, morphological observations, and photograph documentation of the same nerve section are also available.
Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting.
Authors: Tsuru S , Kitani H , Oguchi M , Mashiko M , Zinnaka Y , Shimomura Y
Published: April 2011

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.
Immunocytochemical localization of nerve growth factor: effects of fixation.
Authors: Hazen-Martin D J , Simson J A
Published: April 2011

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.
Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture.
Authors: Krause A W , Carley W W , Webb W W
Published: April 2011

Erythrosin B and trypan blue are tested and compared for their effectiveness as vital exclusion stains for mammalian cells in monolayer culture. Both stains are supposed to mark cells that have lost membrane integrity. Fluorescein diacetate (FDA), an efficient vital inclusion stain, is used as a control, as it marks cells retaining membrane integrity. Erythrosin B and FDA are used as fluorescent dyes, whereas trypan blue colors via light absorption. The effectiveness of both vital exclusion stains is assayed by their ability to stain a high percentage of monolayer cells exposed to treatments lethal to an entire cell population. Two types of lethal treatment, severe heat and metabolic poison, are employed. Erythrosin B stains all monolayer cells immediately after complete lethal treatment. Trypan blue optimally stains only about 60% of monolayer cells. Cell staining by erythrosin B and by FDA are found to be mutually exclusive. This result demonstrates the coincidence of viability indications by erythrosin B and FDA and thus confirms the reliability of both viability stains as they probe membrane permeability via independent mechanisms. This study shows that erythrosin B is an effective, nontoxic, and convenient fluorescent vital exclusion dye for three mammalian cell lines in monolayer culture, but tends to disqualify trypan blue for this application.
Human colonic substance P-producing cells are a separate population from the serotonin-producing enterochromaffin cells.
Authors: Sokolski K N , Lechago J
Published: April 2011

Human colonic mucosa was immunostained with antibodies against substance P to identify the endocrine cells containing this peptide in the mucosal glands. Dual immunohistochemical and histochemical studies were also carried out to determine whether these cells are enterochromaffin cells and contain serotonin as claimed in the literature. The results obtained indicate that the normal human colonic substance P-producing cells are not argentaffin cells, nor do they contain serotonin. In addition, they are also negative both for several silver and for other techniques commonly used to identify digestive endocrine cells. They are positive, however, with the argyrophilic technique of Churukian-Schenk. It is concluded, therefore, that the substance P-producing cells of the human colonic mucosa are not a subpopulation of the enterochromaffin cells, but constitute a distinct and independent cell type.
Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections.
Authors: Lucocq J M , Roth J
Published: April 2011

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.
Synthetic neoglycoproteins: a class of regents for detection of sugar-recognizing substances.
Authors: Kataoka M , Tavassoli M
Published: April 2011

Specific interactions between proteins and sugars have recently been emphasized in many biological systems. To detect sugar-recognizing substances, known as lectin-like substances or endogenous lectins, we describe a method in which various sugars were covalently bound to a carrier protein such as albumin. This neoglycoprotein was stable at -20 degrees C for a period of 6 months. It was conjugated to various cytochemical markers (125I, fluorescein isothiocyanate, colloidal gold, or latex minibead). Detection of the marker then indicates the presence of the sugar-binding protein. Control experiments in the presence of unlabeled neoglycoprotein or specific sugar indicated the specificity of the reaction. This method was used to analyze the kinetics of binding for a mannose-recognition system in the mouse peritoneal macrophages. The data obtained were in agreement with those previously reported. The method can be used for detection of other sugar-recognizing systems as virtually every simple sugar can be bound to a carrier protein to produce these neoglycoproteins. Some of the consideration required for successful production of these reagents are discussed. These synthetic neoglycoproteins are useful in studying the distribution and kinetics of sugar-recognizing systems and may help to further our understanding of this rapidly developing area.
Determination of protein loss during aqueous and phase partition fixation using formalin and glutaraldehyde.
Authors: Mays E T , Feldhoff R C , Nettleton G S
Published: April 2011

In phase partition fixation tissue is immersed in an organic solvent at equilibrium with an aqueous phase containing a fixing agent. By using radioisotope labeling techniques the effects of phase partition fixation on protein retention during fixation of tissue with formalin and glutaraldehyde have been determined and compared with those of standard aqueous fixation using these fixatives. It has been shown that retention of protein in tissue during phase partition fixation was as good or better than during aqueous fixation. Improved retention provides further evidence that phase partition fixation may be a useful alternative to aqueous fixation.
Histochemical evaluation of glycosaminoglycan deposition in the skin.
Authors: Kupchella C E , Matsuoka L Y , Bryan B , Wortsman J , Dietrich J G
Published: April 2011

Histologic demonstration of glycosaminoglycan (GAG) deposition in the skin has been based on the use of either colloidal iron or alcian blue. To define the best technique for the determination of skin GAG content we undertook a prospective study comparing the two stains and evaluating the use of cetylpyridinium chloride (CPC) to enhance fixation. Slides were prepared from skin biopsies obtained from five patients with cutaneous mucinoses. The preparations were coded and examined by three observers. Colloidal iron staining gave a higher intensity for GAG deposits in papillary and reticular dermis. Digestion by specific enzymes identified similar GAGs with either colloidal iron, or alcian blue; however, colloidal iron made GAGs more obvious, partly due to the contrast afforded by the yellow background stain. The addition of CPC to the fixative appreciably enhanced GAG fixation without interfering with the action of enzymes. Experimentally, we confirmed this effect of CPC by determining a pronounced decrease in GAG leakage into the fixative from CPC treated human umbilical cord. We conclude that the combination of CPC fixation and colloidal iron staining gives the best definition of skin GAGs in clinical specimens.
Calcium binding sites in plasmodia of Physarum polycephalum as revealed by the pyroantimonate technique.
Authors: Achenbach F , Achenbach U , Kessler D
Published: April 2011

Plasmodia of the acellular slime mold, Physarum polycephalum, were treated with an osmium tetroxide fixative containing potassium pyroantimonate to precipitate calcium and thereby localize calcium binding sites and sites of increased calcium concentration. Dense calcium pyroantimonate precipitates were detected within the nucleoli. The distribution of these precipitates during interphase and mitosis coincides with the distribution of the unique minichromosomes in Physarum, i.e., the numerous short pieces of extrachromosomal nucleolar chromatin containing segments of amplified DNA coding for ribosomal RNA. Calcium pyroantimonate precipitates were present as frequent dense granules in the mitochondrial matrix and as fine precipitates in the mitochondrial nucleoid. Large calcium-containing precipitates were seen within cytoplasmic vacuoles, confirming reports by others. In addition, we have identified calcium binding sites along the cytoplasmic surface of the plasma membrane. The distribution of calcium within the plasmodium is discussed in relation to the assembly of the mitotic spindle and the regulation of cell motility.
Immunocytochemical localization of lysozyme and alpha-1-antichymotrypsin in the term human placenta: an attempt to characterize the Hofbauer cell.
Authors: Braunhut S J , Blanc W A , Ramanarayanan M , Marboe C , Mesa-Tejada R
Published: April 2011

The intracellular localization of lysozyme (LSZ) and alpha-1-antichymotrypsin (A1Ac), glycoproteins associated with macrophages, was used to confirm the monocytic lineage of the Hofbauer cell and to assess the maturity of its macrophage function. The peroxidase-labeled antigen method was used to localize these proteins, as well as immunoglobulins, light chains, and albumin, in Bouin-fixed, paraffin-embedded sections of 24 normal term placentas. The demonstration of the latter substances was used as an indication of passive diffusion or phagocytosis of serum proteins resulting in intracellular localization unrelated to synthesis. In all the placentas examined a strong cytoplasmic reaction for A1Ac was seen in the Hofbauer cells. The same cells on adjacent sections did not stain for LSZ, while the occasional maternal macrophage and numerous polymorphonuclear leukocytes in the intervillous spaces gave a positive reaction. The detection of A1Ac supports the contention that these cells are macrophages, previously suggested by their phagocytic capability and the demonstration of Fc receptors and nonspecific esterases. Since they do not appear to contain LSZ, a bactericidal enzyme, we propose that these cells are not fully differentiated macrophages, and the lack of this enzyme may have some relevance to the pathogenesis of certain placental infections.
Purification of mouse brain (Na+ + K+)-ATPase catalytic unit, characterization of antiserum, and immunocytochemical localization in cerebellum, choroid plexus, and kidney.
Authors: Siegel G J , Holm C , Schreiber J H , Desmond T , Ernst S A
Published: April 2011

The denatured catalytic polypeptide of mouse brain (Na+ + K+)-adenosine triphosphatase(ATPase) was separated from microsomal membranes on polyacrylamide gels and used as an immunogen. The antiserum, characterized by immunoblots, recognizes the polypeptide corresponding to the catalytic unit in various fractions of mouse brain and cross-reacts with the catalytic unit from lamb kidney, duck salt gland, and electroplax. The same polypeptide in brain and salt gland is recognized by antiserum raised against purified lamb kidney enzyme. Light microscopy was performed with the peroxidase-conjugated second antibody method. In mouse cerebellum, immunochemical staining outlines Purkinje cell and granule cell perikarya. Intense activity is associated with regions of high synaptic content including the pericellular basket meshes and preaxonal regions of Purkinje cells and the glomeruli in the granular layer. In the molecular layer, the neuropil is diffusely reactive with distinct vertically oriented processes evident. White matter exhibits light stain deposition. Choroid plexus presents abundant reaction product only at ependymal apical surfaces, while the ependymal lining of the fourth ventricle displays little or no immunoreactivity. Specificity of the antiserum was demonstrated further in mouse kidney where staining conforms to the well-characterized localization of the enzyme along basolateral surfaces of cortical and medullary tubules. The biochemical and immunocytochemical data show the efficacy of generating antisera to brain (Na+ + K+)-ATPase using catalytic polypeptide as an immunogen.
Staining of the calcification front in human bone using contrasting fluorochromes in vitro.
Authors: Aaron J E , Makins N B , Francis R M , Peacock M
Published: April 2011

Histological studies on bone from the human iliac crest have suggested that if the customary techniques of labeling in vivo with certain fluorochromes such as tetracycline are replaced or augmented by simple whole tissue staining in vitro using the same reagents, differences in the pattern of extracellular fluorescence between normal and pathological states are retained. In particular, the association of the fluorescence with the calcification front in stained examples is closely comparable to similar examples that have been labeled (r = 0.988, p less than 0.001) and to other tinctorial methods (r = 0.891, p less than 0.001). While the stain alone lacks the time lapse of multiple labels, when administered some days after a single label at the time of biopsy, it provides an effective second marker and a measure of mineralization rate that does not differ significantly from that using double labels. Moreover, since the problems of toxicity are avoided by staining, a range of contrasting fluorochromes previously restricted to animal studies may now be used in man. In particular, the enhanced color differentiation brought about by the combination of tetracycline label followed by xylenol orange stain improves image resolution and greatly aids interpretation.
Histochemical detection and quantification of macrophages in rhesus and cynomolgus monkey atherosclerotic lesions.
Authors: Davis H R , Vesselinovitch D , Wissler R W
Published: April 2011

A detailed histochemical study of the macrophage involvement during experimental atherogenesis in rhesus and cynomolgus monkeys was performed. Aortic, carotid, and femoral artery lesions were examined in both species after 4, 8, and 12 months of atherogenic diet feeding. Macrophages were identified and quantified in the atherosclerotic lesions using acid lipase, acid esterase, beta-galactosidase, and cytochrome oxidase histochemical procedures. Morphometric quantitation revealed that the cynomolgus monkey arterial lesions were larger and consistently demonstrated a greater number of cells with characteristics of macrophages in the intimal, medial, and adventitial portion of the arteries when compared to the primarily intimal rhesus monkey lesions. Biochemical assays of aortic samples for acid lipase and acid esterase activity also showed consistently higher activities in the cynomolgus samples when compared to the rhesus samples. Average serum cholesterol levels were higher in the cynomolgus monkeys than in the rhesus monkeys, but the differences in the arterial lesions still existed when animals with overlapping cholesterol levels were compared. Macrophages and their associated activities predominated in experimental cynomolgus monkey atherosclerosis when it was compared to the rhesus disease process, which may be an explanation for some of the differences in atherogenesis reported in these two species.
Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study.
Authors: Taylor A N
Published: April 2011

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.
The secretory contents of Paramecium tetraurelia trichocysts: ultrastructural--cytochemical characterization.
Authors: Kersken H , Tiggemann R , Westphal C , Plattner H
Published: April 2011

The secretory contents ("matrix") of Paramecium tetraurelia trichocysts expand by a factor of 4.5 when they undergo a Ca2+-mediated decondensation in the course of exocytosis. This is paralleled by a concomitant increase in the interval of the periodic banding of the matrix from 12 nm to 45-51 nm, which becomes visible with different electron stains for proteins and negatively charged groups. Recent reports of actin in secretory contents led us to investigate its redistribution and artifactual adsorption to the trichocyst contents upon their expansion. To visualize this effect we used peroxidase-labeled F(ab) fragments from an IgG directed against Paramecium actin, a DNAase I-gold complex, and the induction of F-actin polymerization. The trichocysts were analyzed in situ as well as after isolation by density-gradient centrifugation. Additionally, in response to current reports in the literature, we reanalyzed trichocyst contents for any possible presence of calmodulin. We applied three independent in situ methods for this: autofluorescence after trifluoperazine affinity labeling, calmodulin-fluorescence affinity labeling, and an electron microscopic immunocytochemical method. All three methods failed to reveal any significant labeling of structurally intact trichocysts in situ, although we also showed that discharged trichocysts avidly adsorb calmodulin from the culture medium. From the present data we conclude that the decondensation of trichocysts during exocytosis is mediated by a sudden conformational rearrangement of secretory proteins in the trichocyst contents, without the involvement of any other regulatory or contractile proteins, which occur only in the cytoplasm. Trichocyst contents are not significantly--if at all--glycosylated.
The effects of lanthanum on the ultrastructure of hypertrophic chondrocytes and the localization of lanthanum precipitates in condylar cartilages of rats fed on normal and rachitogenic diets.
Authors: Morris D C , Appleton J
Published: April 2011

Ionic lanthanum was used to examine the distribution of calcium-binding sites in the condylar cartilages of rats that had been fed on normal and/or vitamin D-deficient rachitogenic diets for 4 weeks. Certain specific changes in the ultrastructure of the cartilage were attributed to the presence of the trivalent cation in the fixation medium. Cartilages from the rachitic animals showed a marked reduction in the quantity of lanthanum deposition on the outer surface of the chondrocytes and in the extracellular matrix. Returning rachitic rats to the control diet resulted in a corresponding return to a normal ultrastructural distribution of lanthanum in the condylar cartilages. No intracellular lanthanum deposits were observed. The results suggest that a vitamin D-dependent calcium-binding component may be an integral part of the chondrocyte plasma membrane structure.
The oxidative generation of sulfonic acid groups in neuromelanin and lipofuscin in the human brain.
Authors: Barden H
Published: April 2011

Sulfonic acid groups were oxidatively generated in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Exposure of these oxidized, intraneuronal pigments to low pH Alcian blue or aldehyde fuchsin demonstrated an intensity of staining that related to the type of oxidant and the conditions of its use. Utilization of the following oxidants generated increasingly strong staining reactions as signified by the following sequence; periodic acid under mild conditions, bromine in carbon tetrachloride, hydrogen peroxide, periodic acid under drastic conditions, potassium permanganate followed by oxalic acid, hydrogen peroxide followed by bromine in carbon tetrachloride, potassium permanganate followed by metabisulfite or bisulfite, and performic acid. Neither Alcian blue nor aldehyde fuchsin revealed oxidatively generated aldehyde as judged by 1) their failure or near failure to stain inferior olive lipofuscin following mildly applied periodic acid, and 2) the increase in staining intensity, from moderate to strong, displayed by the soluble lipid-free lipofuscin component of neuromelanin and by inferior olive lipofuscin when potassium permanganate was followed by a rinse with metabisulfite or bisulfite in place of one with oxalic acid.
Localization of lysosomal and peroxisomal enzymes in the specific granules of rat intestinal eosinophil leukocytes revealed by immunoelectron microscopic techniques.
Authors: Yokota S , Tsuji H , Kato K
Published: April 2011

Immunoelectron microscopic localization of lysosomal and peroxisomal enzymes in the eosinophil leukocytes of rat intestinal mucosa was studied by use of rabbit antibodies to the enzymes coupled to protein A-gold complex. Gold particle labeling for the lysosomal enzymes, beta-glucuronidase and cathepsin D, was present on specific granules, with a heavy concentration on their paracrystalline cores. The peroxisomal enzymes, acyl-CoA oxidase and catalase, were also found on these granules. The double labeling procedures using two different combination of anti-acyl-CoA oxidase and anti-beta-glucuronidase or anti-catalase and anti-cathepsin D revealed that these enzymes were simultaneously present in specific granules of the intestinal eosinophils. Quantitative analysis of the labeling on subcellular compartments confirmed that all enzymes examined are significantly localized within specific granules and that there is no significant labeling on other compartments such as the nucleus and cytoplasm. In the control sections incubated with an immunoglobulin G fraction from nonimmunized rabbits, no specific labeling was seen on the granules or other organelles. These findings indicate that enzymes which previously have been identified in some organs as lysosomal and in others as peroxisomal can be found together in eosinophil granules.
Ultrastructural localization of fibronectin to different anatomic structures of human skin.
Authors: Fleischmajer R , Timpl R
Published: April 2011

Fibronectin was localized in fetal and adult human skin with affinity-purified antibodies, using a ferritin staining technique. The most common observation was a close association of this fibronectin with thin and thick cross-striated fibrils containing collagens types I and III. Deposits of fibronectin occurred in discrete spots, with some regular distribution, in agreement with a major binding site for this protein on collagen. Fibronectin was also detectable at the periphery of elastic fibrils and in amorphous, non-fibrillar regions of skin. The latter pattern included a close pericellular localization, indicating interactions between fibronectin and plasma membranes. Discrete deposits of fibronectin were also found on the lamina lucida of the basement membrane of the dermal-epidermal junction and around small blood vessels. This widespread distribution of fibronectin suggests that it has a multitude of biologic functions in situ.
Comparison of protein A-gold and ferritin immunoelectron microscopy of Semliki Forest virus in mouse brain using a rapid processing technique.
Authors: Evans N R , Webb H E
Published: April 2011

Processing tissue for transmission electron microscopy by standard laboratory methods can take two to three days. This makes the development of new techniques time consuming and generally restricts the use of the electron microscope in routine diagnostic work. The possibility of viewing tissue with the electron microscope five hours after sampling using rapid processing techniques is presented. The morphology of the tissue appears undamaged with cell and organelle ultrastructures being readily recognized, as is the presence of virus and its replicating stages. When combined with immunoelectron microscopy a rapid labeling protocol is possible. We have used the technique to develop protein A-gold (6 and 16 nm particles) and ferritin immunoelectron microscopic techniques to demonstrate viral antigens in brain cell cultures and brain tissue from mice infected with Semliki Forest virus.
Species-specific second antibodies reduce spurious staining in immunocytochemistry.
Authors: Houser C R , Barber R P , Crawford G D , Matthews D A , Phelps P E , Salvaterra P M , Vaughn J E
Published: April 2011

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.
Cytokinetics of subpopulations in mixed heteroploid tumors by television imaging. I. Deconvolution of the S-phase DNA ploidy composition. II. Analysis of the S-phase emptying profile of ploidy subpopulations.
Authors: Sklarew R J
Published: April 2011

A scheme has been developed for deciphering the cell cycle time parameters of cell subpopulations that differ in their DNA ploidy level and that coexist in mixed heteroploid tumors. The S-phase analysis is presented. The approach is coupled to an automated imaging methodology for simultaneous determination of the Feulgen-stained DNA content and grain count of 3H-thymidine-labeled cells in autoradiographs (Sklarew RJ: J Histochem Cytochem 30:35, 30:49, 1982). The experimental designs involve 3H- and 14C-thymidine double labeling and Colcemid incubation. The deconvolution of the S-phase ploidy composition is illustrated in rat sarcoma cultures comprising four major ploidy subpopulations; with G-2 and mitotic DNA contents of approximately 4C, 8C, 16C, and 32C. The components were identified by their DNA ploidy level, and their S frequencies and labeling indices were obtained. A scheme is also developed and validated for obtaining the S-phase emptying profile of component ploidy subpopulations, and their cell flux at the S/G-2 and G-2/mitosis phase boundaries. In the sarcoma cultures S mobility was found to decrease with increasing DNA ploidy level over the entire ploidy range.
Acid, neutral, and alkaline phosphatases in developing rat oral mucosa.
Authors: Sjögren S
Published: April 2011

Developing rat oral mucosa contains high activities of acid (AcidPase), neutral (adenosine monophosphatase AMPase), and alkaline (AlkPase) phosphatases. This study is concerned with a detailed analysis of the distribution of these enzymes in freeze-dried sections of oral mucosa. The sections were incubated for AcidPase. AMPase, and AlkPase in the presence of certain inhibiting agents. Four AcidPases, three AMPases, and two AlkPases were identified in the oral mucosa. Six of them were found in the epithelium and three in the connective tissue. Three of the epithelial phosphatases were unique in the sense that they were found only in junction mucosa epithelium. The function of these enzymes may be related to the maintenance of the epithelial cell attachment in an area subjected to stretching, possibly by affecting the periphery of the cells.
Application of immunoelectron microscopy for identification of T lymphocyte subsets and HLA DR antigen positive cells in tissue.
Authors: Dienes H P , Essling K , John H D
Published: April 2011

The identification of T lymphocyte subsets by means of monoclonal antisera in tissue has so far been restricted to light microscopic observations on cryostat sections, since the conventional fixatives as formaldehyde, B5, or glutaraldehyde seem to denature the surface antigens. Applying a mild fixative, periodate-lysine-paraformaldehyde (PLP), we were able to demonstrate T cell subsets on the electron microscopic level in human tonsils with minimal loss of antigenicity of the surface markers Leu 1, OKT4, OKT8, and HLA DR. The preservation of the tissue could be compared favorably to fixation with glutaraldehyde, and provided an ultrastructural basis to distinguish helper and suppressor cells by morphological features as well.