bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of fluorescent compounds in the firefly light organ.
Authors: Smalley K N , Tarwater D E , Davidson T L
Published: April 2011

Two fluorescent materials have been localized in the adult firefly light organ by fluorescence microscopy. One of these is located in photocyte granules, has a maximum emission between 510 and 540 nm, is more fluorescent in basic than acidic solution, and is unstable in ultraviolet light, phosphomolybdic acid, and potassium permanganate. It is thought to be luciferin. The fluorescence of this material is very dim in untreated fireflies but increases substantially following sustained light emission induced by synephrine or prolonged electrical stimulation. It is suggested that the luciferin of untreated animals is bound in the granules and that binding suppresses its fluorescence. The second fluorescent material is located in the dorsal layer of the light organ, particularly in the cells bordering on the photogenic layer. This material has a maximum emission between 510 and 520 nm, is relatively stable in ultraviolet light, and rapidly disappears when light organs are exposed to water. Its identity and function are unknown.
9-AAP, a fluorescent beta-adrenergic antagonist, enters the hamster sperm acrosome in a manner inconsistent with binding to beta-adrenergic receptors.
Authors: Cornett L E , Meizel S
Published: April 2011

We have previously shown that the hamster sperm acrosome reaction, an event essential for fertilization, is stimulated by beta-adrenergic agonists and inhibited by beta-adrenergic antagonists. In the present report, we describe attempts to use (+/-)-9-aminoacridylpropranolol (9-AAP), a fluorescent derivative of a potent beta-adrenergic antagonist, to microscopically detect beta-adrenergic receptors on cauda epididymal hamster spermatozoa in vitro. 9-AAP binding to washed hamster spermatozoa was localized primarily in the acrosomal region, but we were unable to consistently displace the 9-AAP with the biologically active (-)-stereoisomers of several beta-adrenertic agonists and antagonists. Such displacement is necessary in order to separate binding to receptors from "nonspecific binding." Thus we did not detect sperm beta-adrenertic receptors by this method. Failure to detect the receptors with 9-AAP may be due to their presence in numbers too low for detection by this compound or to the masking of the receptors in these uncapacitated sperm. However, we could displace 9-AAP with either 5.0 mM NH4Cl or 1.2 microgram/ml nigericin, compounds capable of discharging pH gradients across cell membranes. These compounds have also been previously reported to displace the fluorescent portion of 9-AAP, 9-aminoacridine from the acrosome by such mechanism. The present results suggest that 9-AAP fluorescence does not always represent binding to beta-adrenergic receptors or "nonspecific binding," but may also represent the concentration of 9-AAP in acidic compartments within a cell.
Carbonic anhydrase in rat liver and rabbit skeletal muscle: further evidence for the specificity of the histochemical cobalt-phosphate method of Hansson.
Authors: Lönnerholm G
Published: April 2011

Rat liver and rabbit skeletal muscle were studied by Hansson's method for histochemical demonstration of carbonic anhydrase activity. In histochemical model experiments purified male rat liver carbonic anhydrase was much more resistant to acetazolamide than rat erythrocyte carbonic anhydrase. Male rat liver slices showed cytoplasmic staining, which was about 1000 times more resistant to acetazolamide and ethoxzolamide than that of female rat liver or erthyrocytes of either sex. Rabbit skeletal muscle slices showed staining at the sarcolemma of all fibers, whereas the staining of the sarcoplasm varied. The walls of capillaries situated within the muscle bundles were intensely stained. The sarcoplasmic staining of a certain number of fibers was at least 1000 times less sensitive to acetazolamide than the other staining. These findings, which are in good agreement with biochemical data, show that the sulfonamides inhibit histochemical staining in a specific way. This is strong evidence for the specificity of the method.
Ultrastruct and tartrate-resistant acid phosphatase localization in a T-cell hairy-cell leukemia cell line.
Authors: Quan S G , Poolsawat S S , Golde D W
Published: April 2011

Hairy-cell leukemia is characterized clinically in splenomegaly and pancytopenia and pathologically by the proliferation in hematopoietic tissue of cells containing the tartrate-resistant isozyme 5 of acid phosphatase. We have described a patient with a T-lymphocyte variant of this disease. A permanent cell line obtained from the spleen of this patient has the biological and enzymatic characteristics of the fresh leukemic cells. We have used this line to study the surface morphology, ultrastructure, and ultrastructural localization of acid phosphatase in defined T-lymphoid hairy cells. The surface of the cells of the permanent line was smooth but many hair-like projections appeared after exposure to phytohemagglutinin (PHA). There was little acid phosphatase reaction produce visualized when beta-glycerophosphate was used as a substrate. With sodium haphthol AS-BI phosphoric acid heavy deposits were seen in the perinuclear membrane, mitochondria, and rough endoplasmic reticulum. Exposure to PHA and pokeweed mitogen resulted in increased reaction product, suggesting increased enzyme synthesis. Tartrate-resistant acid phosphatase was localized in the same organelles.
Optical errors in scanning stage absorbance cytophotometry. II. Application of correction factors for residual distributional error, glare and diffraction error in practical cytophotometry.
Authors: Duijndam W A , Van Duijn P , Riddersma S H
Published: April 2011

In a preceding article theoretical methods were derived for correcting the integrated absorbance values of microscopic objects determined with a scanning stage cytophotometer for the systematical erros due to residual distributional error, diffraction error, and glare error. For an experimental investigation of these results, the local apparant transmission at the center of an opaque particle was determined and this value used as a measure for the substage glare. By sufficient reduction of the size of the illuminated field, this substage glare could be kept below 1% in our scanning stage cytophotometer. The magnitude of the diffraction error was experimentally approached by comparing the values found for the integrated absorbance of the same amount of chromophore, dispersed over two different areas by crushing or centrifugation. After appropriate correction for the residual distributional error, the remaining difference was ascribed to the diffraction error, caused by diffraction at the edges of the object, and to the glare error, present all over the measured area. The local borderline corrections found necessary to obtain the best matching corrected integrated absorbance values were between 3 and 5%, in good agreement with the value derived theoretically. The importance of these corrections for the determination of the integrated absorbance of common biological objects is discussed.
Ultrastructural localization of tubulin in cultured fibroblasts.
Authors: Willingham M C , Yamada S S , Pastan I
Published: April 2011

Antibodies to barin tubulin were created in rabbits and used to localize tubulin in cultured fibroblasts. Cells were fixed and permeabilized using the EGS procedure (Willingham et al: J Cell Biol 79:256a, 1978). Tubulin was localized and quantified using a ferritin-bridge labeling method. Antibody localization was generally confined to morphologically identifiable microtubules, with a smaller concentration in a diffuse cytoplasmic distribution. Following treatment with colchicine, morphologically identifiable microtubules disappeared and the amount of diffuse localization in the cytosol increased. However, a significant concentration of tubulin was found associated with the microfilament mat under the plasma membrane after colchicine treatment, whereas in untreated cells, very little tubulin was found in the microfilament mat. These results demonstrate that tubulin is a component of microtubules and that colchicine treatment results in an association of this tubulin with microfilaments.
Immunocytochemical localization of the posterior lobe hormones and of their carrier proteins.
Authors: Vandesande F , Dierickx K , Goossens N
Published: April 2011

Serial sections of vertebrate hypothalami were stained with the immunocytochemical peroxidase-antiperoxidase method. In addition to the single staining method, our double staining method was used, which enabled us to visualize two tissue antigens in single tissue sections. In both staining methods, differentially adsorbed antineurohypophysial hormone sera, anti-somatostatin serum and anti-bovine neurophysin sera were used. The results confirm the one hormone, one neuron hypothesis.
High content of tyrosine and arginine in peptides and proteins from the growth hormone producing cells of the adenohypophysis.
Authors: Ekman R , Håkanson R , Sundler F , Thorell J
Published: April 2011

Fluorescence histochemistry has demonstrated an abundance of tyrosine and arginine residues in the growth hormone (GH) producing cells of the pituitary of several mammals. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. One granular fraction, rich in GH, was found to contain proteins or peptides rich in tyrosine and arginine. Gel chromatography of the acid-soluble components from sedimented pituitary granules revealed that the arginine- and tyrosine-rich material was heterogeneous. The arginine-containing peptides and proteins could be separated into several peaks with molecular weights from 5000-10,000 and higher. The tyrosine-containing material comprised one peptide with a molecular weight of 5000-10,000 and another much smaller peptide. Since GH itself is not excessively rich in either tyrosine or arginine, the tyrosine- and arginine-containing proteins or peptides probably constitute as yet unidentified granular components, distinct from GH itself.
Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study.
Authors: Cocchia D , Michetti F
Published: April 2011

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.
Immunohistochemical localization of kallikrein in human pancreas and salivary glands.
Authors: Orstavik T B , Brandtzaeg P , Nustad K , Pierce J V
Published: April 2011

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.
Cytochemical study of concanavalin A binding sites and their mobility in normal, cystic fibrosis, and SV40 transformed human fibroblasts in vitro.
Authors: Yokoyama M , Chang J P , Moller P C
Published: April 2011

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.
Effects of fuchsin variants in aldehyde fuchsin staining.
Authors: Lichtenstein S J , Nettleton G S
Published: April 2011

Aldehyde fuchsin stains pancreatic B cell granules, hypophyseal basophils, goblet cell mucins, gastric chief cells, hyaline cartilage, and elastica. Neither the chemical structure of aldehyde fuchsin nor its staining mechanism is known. This study was undertaken to clarify the role of the fuchsin component of aldehyde fuchsin in its staining reaction. The major findings of this investigation include: 1) single N-methylation of the fuchsin molecule abolishes staining of unoxidized pancreatic B cells, although it does not prevent reaction of fuchsin with paraldehyde; 2) aldehyde fuchsin is probably a Schiff base condensation product of pararosaniline and acetaldehyde; 3) a Schiff base structure alone cannot account for aldehyde fuchsin staining of unoxidized pancreatic B cells; 4) a fully potent aldehyde fuchsin is possibly a Tris-Schiff base derivative of pararosaniline.
Histochemical identification of human T lymphocytes from paraffin sections.
Authors: Ranki A , Reitamo S , Konttinen Y T , Häyry P
Published: April 2011

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.
The in situ determination of melting-solidification points of lipid inclusions in fixed cultured cells.
Authors: Malinin G I
Published: April 2011

Melting-solidification points of microscopic lipid inclusions in fetal human kidney cultures were determined visually by gradual heating of formol-fixed cell monolayers while immersed in dilute aqueous solutions of Nile blue A. Intracellular lipid inclusions stained red when their melting point was reached and reverted to blue color at the solidification temperature. The melting-solidification points of intracellular lipid inclusions in kidney cells were determined to fall within 19-25, 40-50, and 60-65 degrees C range.
Effect of glutaraldehyde and decalcifying agents on acid phosphomonoester hydrolase activity in the enamel organ of the rat incisor: a biochemical study comparing enamel organ with liver.
Authors: Smith C E
Published: April 2011

Enamel organs were dissected from the labial surface of unfixed and glutaraldehyde-fixed rat incisors and assayed biochemically at pH 5.0 for acid phosphomonoester hydrolase activity using cytidine 5'-monophosphate (5'-CMP), beta-glycerophosphate (beta GP), phosphorylcholine (PC), and phosphoserine (PS) as substrates. Whole homogenates from unfixed enamel organs showed substantial enzyme activity toward 5'-CMP and beta GP, but 1/4 and 1/8 less activity toward PC and PS, respectively. Perfusion fixation with 2% glutaraldehyde resulted in a net loss of 80% of the enzyme activity toward each substrate. Lineweaver-Burk plots revealed that the fixative depressed the rate of hydrolysis of substrate (decrease in Vmax) and it also lowered the affinity of enzymes for substrate (increase in KM). Hence, fixed tissue generally required two or three times as much substrate to saturate the enzymes, but less substrate was hydrolyzed, as compared to unfixed tissue. Decalcification of fixed incisors with either formic citric acid, ethylenediaminetetraacetic acid (EDTA), or ethyleneglycoltetacetic acid (EGTA) did not further alter enzyme activity in the enamel organ as determined by Lineweaver-Burk plots, However, EDTA and EGTA were found to increase the susceptibility of fixed enzymes to inhibition by lead ions. This chelator-enhanced lead inhibition was greatest following decalcification with EGTA and using PC as substrate. Similar results were obtained for liver.
Immunocytochemical localization of chemoattractant proteinase in whole human skin, fibroblasts, lymphocytes, and granulocytes.
Authors: Morse M A , Farb R M , Thomas C , Lazarus G S
Published: April 2011

A monospecific antibody to purified human skin chemotactic proteinase was raised in rabbits and shown to produce a single line of identity against crude and purified human proteinase. The antibody was used to localize the proteinase in human fibroblasts, polymorphonuclear leukocytes, lymphocytes, and sections of whole skin. All cells demonstrated particulate extranuclear staining. Whole human skin demonstrated staining of the epidermis, especially in the granular layer; particulate staining was also found in the dermis. Staining could be blocked by preincubation of the rabbit anti-human neutral proteinase with purified proteinase antigen. These data indicate that neutral proteinase is located in a number of different human cells in a particulate distribution.
Sensitivity and specificity of virion and cell surface labeling using the unlabeled antibody-hemocyanin bridge method.
Authors: Gonda M A , Gregg M , Elser J E , Hsu K C
Published: April 2011

The sensitivity and specificity of the unlabeled antibody-hemocyanin bridge method for immunoelectron microscopic localization of virion and cell surface antigens has been demonstrated using a hyperimmune serum to the Rauscher murine leukemia virus structural envelope glycoprotein (gp 70). The technique localized the gp70 on the viral envelope and on the infected cell surface at dilutions of greater than 10(-3) for the primary antiviral serum. Little or no nonspecific binding of hemocyanin was detected in control experiments using high concentrations of normal nonreacting or adequately absorbed antiviral primary sera and excess antibody bridge and marker; thus, the system is highly specific. Furthermore, sensitivity can be increased approximately fivefold by marked amplification steps whereby a specimen fixing only a slight amount of hemocyanin can be subsequently treated with antihemocyanin antibody, followed by hemocyanin.
Technical aspects of lymphoma immunohistology.
Authors: Mason D Y , Biberfeld P
Published: April 2011

Immunohistological techniques (i.e., based on tissue sections) for the study of human lymphoma have been developed in recent years as alternatives to immunocytochemical methods (based on cell suspensions). These techniques not only allow the important architectural features of the lymphoma sample to be preserved, but are also more convenient to use in that tissues can be processed rapidly and studied at leisure. The relative advantages and disadvantages of frozen versus fixed embedded tissue, and of immunofluorescence versus immunoenzymatic methods are reviewed, and technical aspects of proteolytic digestion (as a technique for enhancing immunohistochemical labeling) are discussed. This review concludes with a consideration of the problems encountered in the interpretation of immunohistochemical labeling for lymphoma cell immunoglobulin in paraffin sections.
Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography.
Authors: Bergeron J J , Rachubinski R , Searle N , Borts D , Sikstrom R , Posner B I
Published: April 2011

A tissue-screening survey employing quantitative radioautography was carried out at 2 min after the intravascular injection of 125I-insulin into laboratory rats. The results revealed a substantial binding of insulin to cells forming the proximal convoluted tubule in kidney, hepatocytes of liver, acinar cells of the pancreas, parenchymal cells of the adrenal cortex and medulla, and epithelial cells of the gastrointestinal tract. Control experiments indicated that this binding was due to a specific interaction with the insulin receptor, except in the case of kidney where the binding was shown to be nonspecific. Although the major target for insulin action (liver) clearly demonstrated specific insulin binding, several other classical targets (adipocytes, skeletal, cardiac, and smooth muscle cells) showed no specific 125I-insulin binding and therefore indicated the limits of sensitivity of the in vivo radioautographic method. Nevertheless, the working hypothesis of a direct correlation of insulin receptor density with insulin action points to the hitherto unemphasized targets of pancreas, adrenal gland, and gastrointestinal tract as major sites of insulin action in the body.
The diversity of the immunohistological staining pattern of Sternberg-Reed cells.
Authors: Poppema S
Published: April 2011

Lymph node tissue of eight cases of Hodgkin's disease of all different subtypes was studied with an immunoperoxidase technique for the detection of immunoglobulin G (IgG), J chain, kappa and lambda light chains, and alpha-1-antitrypsin in different types of Sternberg-Reed cells. It was found that L&H type Sternberg-Reed cells of the nodular lymphocyte predominance type contained IgG, J chain, and one type of light chain per individual cell. It is concluded that these findings indicate that L&H type Sternberg-Reed cells produce IgG and, consequently, are B immunoblasts. Typical and lacunar type Sternberg-Reed cells of mixed cellularity and nodular sclerosis subtypes were found to contain IgG and both types of light chains per individual cell. J chain was absent from these cells are alpha-1-antitrypsin was found in some of them in a paranuclear pattern, comparable to that in histiocytes. It is concluded that these findings exclude the production of IgG by these types of Sternberg-Reed cells and it is suggested that these Sternberg-Reed cells may be related to histiocytes on the basis of the similarity in the staining pattern for alpha-1-antitrypsin.
The nature of the immunoglobulin-containing cells in malignant lymphoma: an immunoperoxidase study.
Authors: Isaacson P , Wright D H , Judd M A , Jones D B , Payne S V
Published: April 2011

Using the immunoperoxidase technique, an attempt has been made to accurately characterize immunoglobulin (Ig)-containing cells in 185 cases of human malignant lymphoma. By applying a variety of antisera Ig synthesizing cells can be distinguished from cells taking up Ig from the environment. The use of thin (1 mu) paraffin sections has permitted detailed comparison to be made between Ig synthesizing cells of follicle center cell lymphomas and those of reactive follicle centers in human tonsils. Using cell pellets, similar comparison has been made with peripheral blood lymphocytes synthesizing Ig following stimulation with pokeweed mitogen. In follicle center cell lymphomas Ig synthesis is a function of cleaved and noncleaved follicle center cells, not plasma cells, and these cells are strikingly similar to Ig synthesizing cells normally present in nonneoplastic reactive follicle centers and the cells that synthesize Ig following pokeweed stimulation of peripheral blood lymphocytes. these results suggest pathways of B-cell maturation different from those commonly proposed and help to clarify certain inconsistencies in the classification of malignant lymphomas.
Detection of T and B cell antigens hybridoma monoclonal antibodies: a biotin-avidin-horseradish peroxidase method.
Authors: Warnke R , Levy R
Published: April 2011

Acetone-fixed frozen sections of reactive lymph node, spleen, three B cell lymphomas, and three T cell lymphomas were studied for the presence of Ia-like antigens and two T cell antigens. Detection of the binding of the hybridoma monoclonal antibodies to these antigens took advantage of the biotin-avidin interaction. The detection method employed a biotin conjugate of goat anti-mouse antibody and an avidin conjugate of horseradish peroxidase. B cell lymphoma cells stained for Ia. The cell lymphomas were shown to be heterogeneous for the expression of the two T cell antigens. these three antigens were generally not detectable after 10% formalin fixation of B-5 fixation. Detection of these antigens and the method employed should prove useful in the immunologic categorization of human lymphomas.
Biologically active polypeptides in submandibular glands.
Authors: Barka T
Published: April 2011

Since the discovery of kallikreins in the submandibular glands in 1963 by Werle and Roden, a great number of biologically active polypeptides has been purified from, or claimed to be present in, the submandibular of the mouse and of other species. In this review, available data on the occurrence, chemical properties, localization, hormonal control, synthesis, secretion, and possible physiologic roles of 25 biologically active factors in mouse submandibular gland are analyzed. In general, these factors are androgen dependent, but not affected by the sex genotype, and are localized in the granular convoluted tubule cells in the gland. They are secreted into the saliva, but are also found in the circulation. Their physiologic roles are largely unknown.
The distribution and functions of the enkephalins.
Authors: Miller R J , Pickel V M
Published: April 2011

The enkephalins are two pentapeptides with pharmacological properties similar to narcotic drugs. These peptides are widely distributed in the central nervous system. The basal ganglia contain particularly high concentrations of enkaphalins. In the caudate nucleus ultrastructural studies show that enkephalin is contained within synaptic vesicles. Similar observations have been made with respect to enkephalin containing neurons in th A2 nucleus of the medulla and locus ceruleus. In the caudate, lower intensity staining for enkephalin can also be seen in the dendrites of enkephalin positive neurons. Staining for enkephalins is also widely distributed outside the central nervous system. The myenteric plexus in particular contains many enkephalinergic fibers and cell bodies. In addition, enkephalin-containing endocrine cells are found in the gastrointestinal mucosa. These endocrine cells also contain serotonin. Enkephalins have also been detected in several sympathetic ganglia. In some cases postganglionic neurons have been shown to contain enkephalin. SIF (small intensely fluorescent) cells and nerve terminals within sympathetic ganglia also show enkephalin positive staining. Many cells of the adrenal medulla show positive staining for enkephalin.
Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence.
Authors: Beauvillain J C , Tramu G
Published: April 2011

In guinea pig median eminences that were fixed with a glutaraldehyde paraformaldehyde mixtures, postfixed with osmium tetroxide, and embedded in araldite, immunocytochemical stainings for luteinizing hormone-releasing hormone (LH-RH), somatostatin, and alpha 17-39 ACTH have been obtained on semithin sections. These positive reactions were obtained after araldite was removed with sodium methoxide and after treatment with hydrogen peroxide. These data suggest that osmium tetraoxide postfixation can be used for the detection of low molecular weight peptides such as LH-RH, somatostatin, and ACTH-like-peptides not only in the median eminence but also in the hypothalamus.
A rapid single step staining technique for DNA analysis by flow microfluorimetry.
Authors: Taylor I W
Published: April 2011

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.
Rapid in situ cytochemical classification of CFU-C colonies in soft agar culture: permanent whole culture preparations.
Authors: Yourno J , Goldberg J , Nelson D A , Williams W J
Published: April 2011

Soft agar culture CFU-C (colony-forming units in culture) are rapidly classified in situ as eosinophil, macrophage, and neutrophil-monocyte types by whole culture staining with luxol fast blue for eosinophil specific granules and acetoorcein for nuclei. Stained colonies may be picked and examined individually as wet mount cover slip preparations, or the agar culture may be air dried and mounted permanently. The whole culture stain has been variously modified for the enzyme markers alpha-naphthyl acetate esterase and peroxidase and for nuclear staining alone with acetoorcein.
Ultrastructural localization of alpha-OH acid oxidase in peroxisomes with the CeCl3 technique.
Authors: Arnold G , Holtzman E
Published: April 2011

alpha-OH acid oxidase activity was demonstrated in peroxisomes of glutaraldehyde-fixed tissues using the CeCl3 cytochemical method. For kidney, alpha-OH butyrate or alpha-OH valerate were used as substrates. These substrates gave much less reaction product in liver peroxisomes. Liver peroxisomes were more reactive with glycolate as substrate. Glycolate gave little or no reaction product in kidney peroxisomes. alpha-keto-glutarate inhibited activity of the enzyme with alpha-OH butyrate as substrate.
Glicentin and gastric inhibitory polypeptide immunoreactivity in endocrine cells of the gut and pancreas.
Authors: Larsson L I , Moody A J
Published: April 2011

The distribution of the postulated glucagon precursor, glicentin, as well as of the gastrointestinal hormone GIP (gastric inhibitory polypeptide), has been studied by immunocytochemistry and radioimmunoassay. Our results show that GIP antisera may contain a population of antibodies recognizing an immunoreactant common to glicentin and GIP. The occurrence of such common immunoreactants makes immunological distinction between the two hormones difficult and may explain previous results indicating that GIP is stored by glucagon cells. The present results indicate that GIP is produced by endocrine cells of the duodenum and jejunem and is absent from the pancreas, stomach, and large intestine. Glicentin-like immunoreactivity is displayed by A cells of the pancreas and by oxyntic A cells of the stomach, as well as by numerous glucagon-like immunoreactant (GLI) cells of the ileum and colon. Use of glucagon ad glicentin antisera of differing specificities indicates that the processing of this putative prohormone differs between A cells and GLI cells. Studies on the ontogeny of pancreatic A cells also reveal differences in the reactivity pattern of glicentin-like immunoreactivity between fetal and adult rats. Ultraimmunocytochemical studies show that glicentin-like immunoreactivity is mainly, stored in the cytoplasmic granules of pancreatic A cells.
Antibody labeling of thin sections of skeletal muscle with specific antibodies: a comparison of bovine serum albumin (BSA) embedding and ultracryomicrotomy.
Authors: Griffiths G W , Jockusch B M
Published: April 2011

Bovine serum albumin (BSA) embedding and ultracryomicrotomy were used to prepare thin sections of glutaraldehyde-fixed skeletal muscle; these were then treated with antibodies against alpha-actinin, myosin, and actin. Three criteria were then used to compare these two techniques: 1) The preservation of fine structure; 2) the specificity of labeling with antibodies and 3) the amount of antibody bound to a particular antigen. Fine structure was better preserved using ultracryomicrotomy. Both techniques, under optimal conditions, gave specific labeling of muscle components. The amount of antibody bound was higher for BSA sections than for frozen sections. The conclusion is that, while ultracryomicrotomy gives superior qualitative results, the most reliable quantitative estimates would be obtained by using both methods together. Ultracryomicrotomy has the additional advantage that semithin sections can be visualized by immunofluorescence.
Isolation of mouse bone marrow neutrophils by light scatter and autofluorescence.
Authors: Watt S M , Burgess A W , Metcalf D , Battye F L
Published: April 2011

Murine bone marrow and blood cells have been analyzed and fractionated using an automated FACS II cell sorter. Using visible light scattered in the direction of (0 degrees) and perpendicular to (90 degrees) the laser beam it was possible to enrich for neutrophils (84%), immature myeloid cells (47%), and monocytes (78%). The enrichment for neutrophils was improved to 92% by using the light scattered by ultraviolet laser light (ca.360 nm). The autofluorescence at these wavelengths proved useful for obtaining further enrichment (to 97%). Indeed, three parameter sorting with 0 degrees and 90 degrees light scatter as well as autofluorescence also allowed the separation of lymphocytes (95%) and immature myeloid cells (89%). The same procedures could be applied for the isolation of neutrophils from mouse peripheral blood.
Histochemical reactivity of peanut lectin-horseradish peroxidase conjugate.
Authors: Stoward P J , Spicer S S , Miller R L
Published: April 2011

A peanut lectin-horseradish peroxidase (PL-HRP) conjugate has been applied to histochemical staining of paraffin sections of various mouse organs. The PL-HRP conjugate has selectively reacted with secretory bodies, the Golgi zone, and the apical cell surface in various cell types. Some positive sites, including lingual and tracheal serous glands, Brunner's glands, and the brush border of the proximal straight nephron, contained periodic acid-Schiff (PAS)-positive glycoconjugate with no affinity for basic reagents. The stored secretion in these sites was interpreted as containing neutral glycoprotein with terminal galactose residues which could, in part at least, account for the PAS reactivity. Duodenal goblet cells, which exhibited basophilia attributable to sulfate esters, also bound PL-HRP. As the binding was affected by prior sialidase digestion, the secretory glycoprotein in the duodenal goblet cells was judged to contain oligosaccharides with sulfate esters and terminal galactose uncapped by sialic acid. All sites known from their basophilia to form sialomucin failed to stain with the PL-HRP conjugate, but consistently gained reactivity following sialidase digestion and were inferred, therefore, to possess glycoproteins with oligosaccharide side chains containing subterminal galactose and terminal sialic acid. Lingual mucous glands, known to secrete a mucosubstance with basophilic properties indicative of the presence of sulfate esters but not sialic acid, stained with PL-HRP only after sialidase digestion and, accordingly, were reinterpreted as containing both sulfate esters and terminal galactose-sialic acid dimers. Staining of gastric surface epithelium demonstrated a srongly PAS-reactive neutral glycoprotein, and that of goblet cells in the cecum disclosed PAS-positive sulfated glycoprotein. The latter two sites lacked PL-HRP affinity without or with prior sialidase treatment and apparently possessed neither terminal galactose residues nor galactose-sialic acid dimers. PL-HRP affinity was observed exclusively in the Golgi cisternae of some epithelial cells, thus indicating that galactose occurs transiently as a terminal residue in this site. A few histologic sites, such as pancreatic and gastric zymogen cells and renal tubules, were devoid of both PAS reactivity and basophilia indicative of the presence of complex carbohydrate but stained strongly with the PL-HRP conjugate by means which are not understood. Galactose in the PL-HRP solution blocked or reversed the PL-HRP binding in most of the structures with an affinity for the conjugate, supporting the conclusions that the reagent is specific for galactosyl residues.
Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue.
Authors: Rybicka K
Published: April 2011

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.
A new method for the preparation of metaphase chromosomes for flow analysis.
Authors: Sillar R , Young B D
Published: April 2011

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.
Intracellular localization of actin in cultured fibroblasts by electron microscopic immunocytochemistry.
Authors: Willingham M C , Yamada S S , Davies P J , Rutherford A V , Gallo M G , Pastan I
Published: April 2011

Antibodies to skeletal muscle actin were produced in rabbits and purified by affinity chromatography. Direct labeling of SDS-PAGE gels of whole cell homogenates from mouse fibroblast cells showed that actin was the only protein detected by these antibodies. Using this immunospecific reagent, we localized actin in cultured fibroblasts using the EGS fixation-permeabilization procedure with the ferritin bridge labeling technique. Swiss 3T3-4 mouse fibroblasts were chosen as an example of highly adherent untransformed cells with prominent microfilament bundles, and L929 mouse fibroblasts were chosen as an example of poorly adherent, rounded, transformed cells with prominent microvilli. Using these two cell types, we have characterized the intracellular distribution of action. Actin was only detected in locations in which morphologically recognizable 60 A microfilaments were found. By both fluorescence and electron microscopy, actin was found in surface ruffles, microvilli, microfilament bundles, the microfilament mat, and the leading lamellae of Swiss 3T3 and L929 cells. In addition, actin was found surrounding micropinosomes and macropinosomes. On the other hand, there was no actin detected around the base of coated pits. Morphometric quantitation showed that almost all the actin was localized in microfilamentous structures. Our results suggest that actin has an important role in cell motility and adhesion, and in the endocytosis of pinosomes, but that actin may not be involved in intracellular processes such as saltatory motion of intracellular organelles.
A simple method for the preparation of cell cultures for ultrastructural investigation.
Authors: Kuhn H
Published: April 2011

A method is described that allows investigation of cultured cells at the light or electron microscopical level without changing the culturing conditions or the in situ situation during dehydration or embedding. Fixed and dehydrated cell culture layers were cut into small pieces and separated from the plastic dish by adding propylene oxide. With this simple method we could obtain orientated sections of selected cell groups.
Visualization of nucleolar substructure in cultured human fibroblasts by magnesium-activated adenosine triphosphatase reaction.
Authors: Fox N , Fernandez C , Studzinski G P
Published: April 2011

Discrete sites of adenosine triphosphatase (ATPase) activity were demonstrated within the nucleoli of unfixed cultured human fibroblasts (IMR90, VA13, and AG2804 cells) by an adaptation, for electron microscopic cyto-chemistry, of Wachstein and Meisel's lead nitrate method. The majority of nucleoli contained more than one ATPase-positive region, but the total ATPase-positive material appeared to occupy only a minor portion of the nucleolar volume. These regions were roughly spherical with an irregular contour, and at times appeared to be components of perinucleolar chromatin or to be located adjacent to nucleolar interstices. The distribution of these regions within the nucleolus and their segregation by actinomycin D suggested that the ATPase-positive regions correspond to the fibrillar centers, which represent nucleolar organizer regions. The cytochemically demonstrable nucleolar ATPase was strictly dependent on the presence of divalent cations. Optimal reactions was seen at 5 mM Mg2+, but near optimal activity was obtained with lower concentrations of Mg2+ in the presence of Ca2+. Calcium alone and Mn2+ alone produced suboptimal reaction. Studies with different nucleoside phosphates as reaction substrates showed that the enzyme is specific for adenosine derivatives, ATP and dATP being equally good substrates. Guanosine triphosphate, cytidine triphosphate, uridine triphosphate, and d-thymidine triphosphate were ineffective as substrates, as were nucleoside mono- and diphosphates and other phosphate esters tested. It is suggested that the cytochemical ATPase reaction visualized the regions of the nucleolus in which ribosomal DNA of intranucleolar chromatin is undergoing conformational alterations.
Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunocytochemistry.
Authors: Anderson R E , Gay C V , Schraer H
Published: April 2011

Carbonic anhydrase was localized in the chick embryo chorionic ectoderm at the ultrastructural level by immuno-cytochemistry. Preembedding staining of whole tissue was performed. The enzyme was present in the cytoplasm, on the membranes of apical vesicles, and on the membranes of microvilli in villus cavity cells, cells that may be involved in acid secretion and subsequent dissolution of the egg shell. In sinus covering cells, the enzyme is solely in the cytoplasm. The location of the enzyme in the thin cytoplasmic arms of the sinus covering cells is consistent with the role in nonrespiratory CO2 release.
Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells.
Authors: Ackerman G A , Wolken K W
Published: April 2011

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.
Microperoxisomes in the epithelial cells of the amphibian urinary bladder: an electron microscopic demonstration of catalase and malate synthase.
Authors: Jones R G , Davis W L , Goodman D B
Published: April 2011

All cells that comprise the epithelium of the toad urinary bladder were found to contain small ovoid to tubular membrane-bound bodies with a finely granular matrix. Such organelles were devoid of dense cores (nucleoids). These microperoxisomes reacted positively when incubated for the demonstration of catalase or malate synthase activity. In the toad liver, peroxisomes as well as microperoxisomes were seen. Histochemically, both demonstrated catalase activity; neither showed malate synthase activity. The presence of malate synthase, a glyoxylate cycle enzyme, in toad urinary bladder microperoxisomes may make these latter organelles unique among vertebrates, since malate synthase has been thought to be absent in higher animals.