bluuebunny/crossref_metadata_2025 huggingface dataset:
Coupling of uridine-5'-diphosphate (UDP) formation and nicotinamide adenine dinucleotide (NAD+) reduction for cytochemical localization of glycosyltransferases.
Authors: Matyas G R , Morré D J
Published: April 2011

A technique applicable to the cytochemical localization of glycosyltransferases through a series of coupled enzyme reactions is described. Uridine-5'-diphosphate (UDP) formed by glycosyltransferases is first phosphorylated to uridine-5'-triphosphate (UTP) by nucleoside 5'-diphosphate kinase. The UTP plus exogenous glucose-1-phosphate is converted into UDP-glucose by uridine-5'-diphosphoglucose pyrophosphorylase. UDP-glucose is then oxidized by uridine-5'-diphosphoglucose dehydrogenase to form UDP-glucuronic acid and reduced nicotinamide adenine dinucleotide (NADH). The NADH is utilized by membrane-located NADH-ferricyanide oxidoreductases in the presence of a copper salt to form electron-dense deposits of cupric ferrocyanide (Hatchett's brown). Using this technique, galactosyltransferase has been localized in cisternae (including the central midregions of the cisternae) of Golgi apparatus isolated from rat liver. Reactivity is absent from the cis-most cisternae and membrane elements. The reaction is dependent on UDP-galactose and inhibited by ethylene diaminetetraacetic acid and puromycin. the latter is a known inhibitor of galactosyltransferase of rat liver Golgi apparatus. The reaction is adaptable by varying the sugar nucleotide donor and acceptor to any glycosyltransferase utilizing UDP-sugars (except UDP-glucose). Presently it is restricted to isolated membrane fractions and permeabilized cells due to the need for accessibility of reagents and coupling enzymes.
Quantitative immunohistochemistry: a comparison of microdensitometric analysis of unlabeled antibody peroxidase-antiperoxidase staining and of microfluorometric analysis of indirect fluorescent antibody staining for nicotinamide adenosine dinucleotide phosphate (NADPH)-cytochrome c (P-450) reductase in rat liver.
Authors: Smith M T , Redick J A , Baron J
Published: April 2011

The intralobular distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c (P-450) reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) in rat liver has been investigated by means of two quantitative immunohistochemical techniques: microdensitometric quantitation of unlabeled antibody peroxidase-antiperoxidase staining and microfluorometric analysis of indirect fluorescent antibody staining. Utilizing sheep antiserum elicited against NADPH-cytochrome c (P-450) reductase that had been isolated and purified to apparent homogeneity from rat liver microsomes, the reductase was detected within hepatocytes throughout the liver. However, differences in the intensity of staining of hepatocytes within different regions of the liver lobule were readily apparent after completion of both immunohistochemical staining procedures. These visual findings were verified by microdensitometric and microfluorometric analyses of immunohistochemical staining, both of which revealed that approximately the same degree of staining for NADPH-cytochrome c (P-450) reductase was produced within the centrilobular and midzonal regions of the liver lobule, whereas periportal hepatocytes were stained with significantly less intensity. These results demonstrate that the application of either microdensitometry in conjunction with unlabeled antibody peroxidase-antiperoxidase staining or microfluorometry after indirect fluorescent antibody staining can be used to quantitatively determine the intratissue distributions of antigens.
Lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia.
Authors: Freeman H J
Published: April 2011

Lectins linked to fluorescein were used as carbohydrate probes to examine the goblet cell mucin and epithelial cell surface glycoconjugate alterations in an experimental rodent model of colonic neoplasia induced with parenteral 1,2-dimethylhydrazine dihydrochloride. Lectins derived from Triticum vulgare (WGA), Ricinus communis (RCA1), and Limulus polyphemus (LPA) showed reduced labeling of goblet cell mucin in these tumors, while binding with peanut lectin from Arachis hypogaea (PNA), a lectin ordinarily failing to bind to mucin in normal colon, was positive. In addition, RCA1 and LPA showed increased cell surface labeling of neoplastic epithelial cells. Finally, alterations were observed in lectin binding to "transitional" colonic mucosa adjacent to colonic tumors from carcinogen-treated rats. These findings indicate that significant alterations in both membrane and mucin glycoconjugates occur in colonic tumors and mucosa adjacent to tumors in a chemically induced experimental animal model of human colon cancer.
Inhibition by dithionite and reactivation by iron of the tartrate-resistant acid phosphatase in bone of osteopetrotic (ia) rats.
Authors: Hammarström L E , Anderson T R , Marks S C , Toverud S U
Published: April 2011

The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.
Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. III. Improvements in Feulgen densitometry.
Authors: Sklarew R J
Published: April 2011

A method has been developed for densitometric estimation of the Feulgen-stained DNA content of 3H-labeled nuclei in autoradiographs in conjunction with automated grain counting using a Quantimet Imaging System. Refinements in the methodology are reported which include 1) the incorporation of an Image-Editor Module into the Quantimet module configuration; 2) the optimization of incident illumination based upon evaluation of various light sources; 3) changes in the optical configuration which reduce glare and minimize the level of monitor shading correction; 4) the optimization of scanner sensitivity; and 5) the evaluation of cell-flattening and staining with respect to densitometry resolution and sensitivity. These refinements resulted in a CV of less than 6.4% in the G-1 and G-2 DNA peaks of rat kidney cells in autoradiographs compared to the previous CV of 10.5%, and a G-2 to G-1 ratio of 2.025. For a fixed field position the CV was 5.1% and the replication error less than 1.0%.
The effects of fixation by combination of glutaraldehyde/dimethyl suberimidate. Use of collagen as a model system.
Authors: Tzaphlidou M
Published: April 2011

The fixative behavior of glutaraldehyde/dimethyl suberimidate in combination was studied using reconstituted fibrils of type I calf skin collagen as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results confirm that the structural preservation of collagen obtained using prefixation in glutaraldehyde followed by dimethyl suberimidate fixation is less than when using dimethyl suberimidate and postfixation in glutaraldehyde. Furthermore, glutaraldehyde preserves the structure of collagen to a lesser degree than dimethyl suberimidate. With the combination of the two fixatives, the preservation of fine structure of collagen obtained is less than that when using dimethyl suberimidate alone.
Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) and LHRH-like material in brain. Influence of fixation solution at various pH values.
Authors: Piekut D T
Published: April 2011

The amount and distribution of luteinizing hormone-releasing hormone (LHRH) immunoreactive (ir) material in the hypothalamus, and of a LHRH-like immunostained substance in the rat pineal gland were examined employing Bouin's and Zamboni's fixation solutions at various pH values, and antisera generated against synthetic LHRH. The hypothalamic LHRH immunoreaction product is optimally visualized in this study when the pH value of the fixation solution is basic; conversely, the pineal LHRH-like ir material is seen only with an acidic pH value of the fixation solution. The present study demonstrates that the pH value of the fixation solution contributes substantially to the amount and distribution of some peptides visualized immunocytochemically in the brain.
Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry.
Authors: Chapman C M , Loewenberg J R , Schaller M J , Piechura J E
Published: April 2011

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.
Nonspecific staining of mast cells by avidin-biotin-peroxidase complexes (ABC).
Authors: Bussolati G , Gugliotta P
Published: April 2011

A nonspecific staining of mast cells by avidin-biotinylated peroxidase complexes (ABC) has been observed and related to an ionic binding of the basic residues of avidin (isoelectric point at pH 10) and peroxidase to the sulphate groups of heparin. This affects the correct interpretation of the results of the immuno-peroxidase ABC procedure, especially in mast cells-rich areas such as the lymphoid tissues. The spurious staining can be prevented by using the ABC solution at pH 9.4 (instead of pH 7.6 as usually recommended): this high pH does not affect the previous binding of primary antibodies nor the affinity of avidin to biotin. The modified ABC procedure provides a clear background and a sharp specific staining and can be recommended for routine use.
An immunohistochemical method for the localization of N-acetylserotonin (NAS) in the central nervous system. Description, validation, and application of the technique.
Authors: Pulido O , Brown G , Grota L J
Published: April 2011

Antisera to N-acetylserotonin (NAS) were raised in rabbits by coupling NAS to bovine serum albumin (BSA) through a p-carboxybenzyl (PCB) bridge at the indole N. The specificity and applicability of these antisera in immunohistochemistry is reported. The anti-NAS antiserum and a fluorescein-labeled immunoglobulin were employed to investigate the topographic distribution of immunoreactive NAS (INAS) in the hindbrain (mesencephalon, cerebellum, pons, and medulla oblongata). Positive identification of INAS was confirmed in the granular layer of the cerebellum, the tractus spinalis nervi trigemini and the reticular formation. INAS was also identified in Purkinje cells, cerebellar nuclei, nucleus principalis nervi trigemini, nucleus tractus mesencephali, cochlear and vestibular nuclei, the locus coeruleus, and other brain stem regions. The pattern of INAS distribution is independent of serotonin (5-HT) and norepinephrine (NE), although certain loci could contain both INAS and serotonin or INAS and norepinephrine.
The immunolocalization of copper-zinc superoxide dismutase in canine tissues.
Authors: Thaete L G , Crouch R K , Schulte B A , Spicer S S
Published: April 2011

Copper-zinc superoxide dismutase (CuZn SOD) has been localized in paraffin-embedded sections of formalin-fixed, canine tissues. Staining with the immunoenzyme bridge sequence and the technique utilizing avidin-biotin-peroxidase complex revealed high levels of CuZn SOD in a number of sites. These included hepatocytes, colonic absorptive epithelial cells, ciliated bronchiolar epithelial cells, cells lining the thick ascending limb of Henle's loop in the nephron, endocrine cells producing hormones in pancreatic islets, and neurons in several regions of the central nervous system. The above results are compared with the reported distribution of CuZn SOD as determined by biochemical methods in tissues of various animal species.
Evidence from thiol histochemistry for homology between the Tabby-crinkled syndrome in mice and human ectodermal dysplasia.
Authors: Weeks N L , Blecher S R
Published: April 2011

Mutant mice carrying the sex-linked gene Tabby (Ta), and others carrying crinkled (cr), an autosomal mimic of Ta, were studied histochemically with respect to the sulfhydryl: disulfide ratios in their skin and hair and the SH levels in their tooth germs. As compared to normal controls, the mutant animals demonstrated significantly elevated SH:S-S ratios (skin and hair) and SH levels (tooth germs) in the ectodermal components of these tissues. This finding corresponds with previously reported biochemical data on a form of human ectodermal dysplasia (ED), and therefore supports the hypothesis that these mutations in mice may be homologous to the genes for human ED.
On the Internal Polarity of the Golgi Apparatus with Special Regard to Its Relationship to GERL
Authors: Fujita Hisao , Sawano Fumio
Published: April 2011

The authors emphasized that from the cytochemical standpoint the Golgi apparatus has an internal polarity and that GERL is a part of this organelle.
Ultracytochemical Observations on Intracytoplasmic Lipids by Enzymic Digestive Methods: Histochemical Trials
Authors: Morii Sotokichi
Published: April 2011

The penetration of Rbizopus arrhizus lipase into ethioine fatty livers was examined histochemically. A free-floating, 30 μm thick Vibratome section of the livers perfused with aldehydes was shown to be suitable for ultracytochemical analysis by the lipase digestive method. Accessibility of phospholipids in the outer membrane of erythrocytes to Naja naja phospholipase A2 was observed under a light microscope, and intact red cells were used for the ultracytochemical analysis by the phospholipase digestive method. Ultracytochemical pictures of cytoplasmic lipids in these materials subjected to the enzymatic digestive method are presented.
Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1.
Authors: Morris R J , Barber P C
Published: April 2011

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.
Electron microscopic immunostaining of kallikrein in rat submandibular glands.
Authors: Simson J A , Fenters R , Chao J
Published: April 2011

The subcellular localization of glandular kallikrein in ducts and tubules of the rat submandibular gland was determined using a postembedding immunostaining (peroxidase--antiperoxidase) technique on thin sections of Eponembedded tissue. Kallikrein was found in large granules of granular convoluted tubule cells and in small, apical granules of striated duct cells. It was also present in patchy aggregates along the surface of striated duct cells and intercalated duct cells, but not in granules of the latter. Basal dense bodies (lysosomes?) of granular tubules also stained for kallikrein. Absorption of kallikrein antiserum with rat urinary kallikrein, but not with rat urinary esterase A, abolished the specific immunostaining in these sites.
Ultrastructure and staining properties of aortic microfibrils (oxytalan).
Authors: Goldfischer S , Coltoff-Schiller B , Schwartz E , Blumenfeld O O
Published: April 2011

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.
Intestinal metaplasia and carcinomas of the human stomach: an immunohistological study.
Authors: Nardelli J , Bara J , Rosa B , Burtin P
Published: April 2011

Rabbit immunization with duodenal or colonic high molecular weight components allowed us to distinguish three new M3 antigens associated with intestinal goblet cells; these were denoted as M3SI, M3D, and M3C. Using immunoperoxidase staining, the anti-M3SI serum labeled all goblet cells in the small intestine, and a certain number of them in the colon, mainly in its proximal part. The anti-M3D serum reacted primarily with the goblet cells of the duodenal villosities, and the anti-M3C serum with all goblet cells of the large, but not the small, intestine. Moreover, the M3C antigen was recovered in some goblet cells of the fetal duodenum, in association with the two other markers. Only the M3D and the M3SI antigens were observed in the normal duodenum and intestinal metaplasia (IM) of benign gastric mucosa. In contrast, IM adjacent to gastric carcinomas, whatever their histological type, contained the M3C antigen in addition to M3D and M3SI antigens, and thus showed an antigenic pattern similar to fetal duodenum. Some gastric carcinomas, especially those with intestinal-like differentiation, produced the colonic M3C antigen as the neighboring IM. Thus, these two tissues displayed common differentiation features, which could be related to fetal duodenum rather than to adult colon.
Selective digestion of human metaphase chromosomes by Alu I restriction endonuclease.
Authors: Mezzanotte R , Ferrucci L , Vanni R , Bianchi U
Published: April 2011

Human cytological preparations have been digested with Alu I restriction endonuclease (Endo R Alu I) and subsequently stained with acridine orange. Metaphase chromosomes treated in this way revealed a clear-cut bright/ dull longitudinal differentiation. It has been postulated that these cytological findings could be related to the selective digestion, by Alu I, of all DNA with the exception of some heterochromatic, possibly satellite, fractions.
Ultrastructural immunocytochemical localization of calmodulin in cultured cells.
Authors: Willingham M C , Wehland J , Klee C B , Richert N D , Rutherford A V , Pastan I H
Published: April 2011

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.
Immunocytochemical localization of fatty acid metabolizing heat-stable and heat-labile enoyl-coenzyme A (CoA) hydratases in liver and renal cortex.
Authors: Bendayan M , Reddy M K , Hashimoto T , Reddy J K
Published: April 2011

Two enzymes, the heat-stable and the heat-labile enoyl-coenzyme A (CoA) hydratases, involved in the metabolism of fatty acids were localized in liver and renal cortex using specific antibodies, immunofluorescence, and the protein A-gold immunocytochemical technique. The qualitative and quantitative results have demonstrated that the heat-stable enoyl-CoA hydratase is a mitochondrial membrane-associated protein of hepatocytes and of epithelial cells in proximal and distal renal tubules. The hepatic sinusoidal cells, as well as the endothelial and epithelial cells of the glomeruli, fail to demonstrate any specific labeling. The heat-labile enoyl-CoA hydratase, on the other hand, was detected in the peroxisomal matrix of hepatocyte and proximal tubule epithelial cells. Its distribution was identical to that of catalase. The significance of the differential distribution of peroxisomal and mitochondrial enoyl-CoA hydratases is discussed in relation to their function.
Histochemical studies on lectin binding in reactive lymphoid tissues.
Authors: Hsu S M , Ree H J
Published: April 2011

Using the avidin-biotin-labeled peroxidase complex (ABC) method, the staining reaction of a panel of 12 biotin-labeled lectins was studied in formalin-fixed, paraffin-embedded reactive lymph nodes and tonsils. Varying degrees of lectin binding were observed in lymphoid cells and macrophage-histiocytes with Concanavalin ensiformis (Con A), Lens culinaris (LCA), Phaseolus vulgaris (PHA), Pisum sativum (PSA), Ricinus communis (RCA), and Triticum vulgaris (WGA) agglutinins, but no evidence of binding was observed with Dolichos biflorus (DBA), Bandieraea simplicifolia (BSA), Arachis Hypogaea (PNA), Glycine soja (SBA), Sophora japonica (SJA), and Ulex europaeus (UEA) agglutinins. Three major patterns of binding were seen: the reaction products occurred along the plasma membranes (membranous), were confined to one pole of the cell membrane (cap-like), or were present diffusely in cytoplasm (cytoplasmic). The cells showing membranous and cap-like staining patterns corresponded to the lymphoid cells, as did the cytoplasmic to plasma cell and macrophage-histiocytes. Cap-like staining was observed on the lymphocytes at B and T cell areas with all six lectins. Thus, the presence of cap-like staining may not be useful for discrimination between B and T cells. Membranous staining, in contrast, was limited to lymphocytes of follicles (B cells) with PSA and LCA, and to germinal center cells with PHA, WGA, Con A, and RCA also reacted with the membrane of T-cell. The cytoplasmic staining reaction of macrophage-histiocytes varied markedly from one lectin to the other. Our study indicates that the carbohydrate moiety of the cells retains their binding sites for lectins through routine processing, providing a means of valid retrospective studies. Furthermore, these observations suggest that each lectin, despite its identical inhibitory sugar, should be tested for its unique reaction pattern, which is not predictable from the data derived from cell suspension studies.
Identification of ending containing dopamine and vasopressin in the rat posterior pituitary by a combination of radioautography and immunocytochemistry at the ultrastructural level.
Authors: Pelletier G
Published: April 2011

In order to study the morphological relationship between the dopaminergic and the vasopressinergic endings in the rat posterior pituitary, a combination of radioautography and immunocytochemistry techniques was applied to the same ultrathin section of the posterior pituitary. Immunocytochemical localization (peroxidase-antiperoxidase technique) of vasopressin was first applied, followed by the radioautography detection of [3H] dopamine that had been injected 30 and 60 min before fixation. This double labeling technique has established that the dopaminergic endings that contain both dense core vesicles (60-100 nm in diameter) and small clear vesicles are always found in close proximity to vasopressinergic neurosecretory endings, suggesting an interaction between these two systems. This relatively easy approach should be very useful to study the connections between different neurotransmitter systems in the nervous system.
Transepithelial endoplasmic reticulum in rat proximal convoluted tubule.
Authors: Novikoff A B , Spater H W , Quintana N
Published: April 2011

Inosine 5'-diphosphatase (IDPase) activity was demonstrated cytochemically in the endoplasmic reticulum of rat kidney proximal tubule cells in tissue fixed by perfusion with glutaraldehyde--formaldehyde. Incubation for IDPase activity at pH 7.2 was performed with and without 0.5 mM levamisole, a potent inhibitor of alkaline phosphatase (AlkPase) (M Borgers, J Histochem Cytochem 21:812, 1973). Levamisole treatment of sections eliminated all reaction product in the brush border, but did not affect the IDPase activity the endoplasmic reticulum (ER). The ER appears as a basilar-luminal-oriented transcellular structure, suggesting a possible cellular transport route. This study supports and extends earlier observations made by others that suggest a transport role for the ER in these cells. It also emphasizes the value of thick section cytochemistry.
Immunohistochemical identification of collagenase in carrageenin granuloma.
Authors: Pardo A , Rosenstein I , Montfort I , Pérez-Tamayo R
Published: April 2011

The collagenase present in experimental carrageenin granuloma in the guinea pig has been purified to homogeneity in acrylamide gel electrophoresis by a combination of ammonium sulfate salting out and affinity chromatography on Sepharose 4B--collagen-packed columns. The single protein band thus obtained was used as an antigen to obtain a monospecific antibody in heterologous conditions. Several immunodiffusion, immunoaffinity chromatography, and immunoinhibition tests of the antibody against the specific antigen and various possible serum and tissue contaminants suggested that the antibody was specifically directed against the enzyme protein collagenase. Indirect immunohistochemical staining of carrageenin granulomas, samples at different developmental phases with this specific anti-collagenase antibody, revealed that the specific antigenic protein (the enzyme collagenase) is universally present on the extracellular structures at both the collagen-deposition and the collagen-resorption stages. A hypothesis is proposed to account for these findings, namely, that the enzyme collagenase is bound to its substrate (collagen) under both normal and pathological conditions, and that the critical point of control of collagen degradation must be the activation of the collagen-bound enzyme.
Penetration of immunoreagents in Vibratome-sectioned brain: a light and electron microscopic study.
Authors: Piekut D T , Casey S M
Published: April 2011

Immunocytochemical studies on the localization of peptides at the ultrastructural level have most frequently involved the application of the peroxidase--antiperoxidase (PAP) method of immunocytochemistry and the use of the preembedding or postembedding staining procedures. The present study was designed to determine the depth of penetration of Vibratome tissue sections by immunoreagents used in the preembedding method in which immunostaining of unembedded fixed tissue sections is accomplished prior to tissue dehydration and embedment. Our data indicate that penetration of immunoreagents is restricted to the superficial 8-9 micrometers of a 80-micrometers thick Vibratome tissue section of hypothalamus of brain using antisera generated against arginine vasopressin. The final immunoreaction product visualized in a Vibratome tissue section may reflect only a fraction of the amount of hormone contained within the thickness of the tissue section.
Subcellular localization of dipeptidyl peptidases II and IV in rat and rabbit alveolar macrophages.
Authors: Sannes P L
Published: April 2011

Pulmonary alveolar macrophages, obtained from healthy adult rats and rabbits by lung lavage, were evaluated for the presence of two serine exopeptidases having affinity for penultimate prolyl residues. Dipeptidyl peptidase II was visualized using either lysyl-prolyl or lysyl-alanyl derivatives of 4-methoxy-2-naphthylamine (MNA) as synthetic substrates at pH 5.5 and employing the osmiophilic coupler hexazotized pararosanaline (HXP) for localization. Reaction product was observed in 50-90% of alveolar macrophages and was confined principally to lysosomal structures. Interestingly, not all lysosomes reacted, suggesting that they have a variable but distinct degree of heterogeneity for this protease. Dipeptidyl peptidase IV was localized using glycyl-prolyl-MNA at pH 7.3 and HXP. Reaction product was observed exclusively on the external surface of the plasma membrane of the rat only. Its intensity appeared to be greater along the outer edge of what might be interpreted as the cell's glycocalyx. These proteases, by their localization and known substrate specificity, would be expected to make a significant contribution to the alveolar macrophage's proteolytic capabilities.
Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity.
Authors: Ciocca D R , Adams D J , Bjercke R J , Sledge G W , Edwards D P , Chamness G C , McGuire W L
Published: April 2011

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.
Cobalt uptake and localization of putative calcium binding sites in striated muscle.
Authors: Anderson P A , Josephson R K
Published: April 2011

Frog sartorius muscles were soaked overnight in saline containing 50 mM cobalt and subsequently fixed with or without precipitation of intracellular cobalt by exposure to ammonium sulfide. Treated fibers and appropriate controls were embedded in plastic in the usual manner. Thick sections (3-4 micrometers) of each sample were treated with silver in order to intensify precipitated cobalt. The samples were then resectioned for ultrastructural examination. Fibers exposed to cobalt with or without precipitation had normal ultrastructure; indeed, the transverse lines of the A-zone and M-band appeared to be more obvious after exposure to cobalt than in control samples. Exposure to silver produced numerous silver grains only in those samples that contained precipitated cobalt. The distribution of silver grains after intensification matched expected calcium binding sites. Silver grains were abundant over membranes of the sarcoplasmic reticulum, sarcolemma, mitochondria, and nucleus and rare or absent from filaments and sarcoplasm. This technique appears to be generally useful for locating intracellular calcium binding sites.
Ultrastructural localization of phenothiazines and tetracycline: a new histochemical approach.
Authors: Hoff S F , MacInnis A J
Published: April 2011

To provide high resolution information on the subcellular localization of the phenothiazines and tetracycline, we have developed a new histochemical method that circumvents the difficulties inherent in classical electron microscopic tissue preparatory procedures. Specific and reliable localizations of these drugs were accomplished by their rapid precipitation with phosphotungstic acid (PTA) at pH 7. A cell suspension of Ehrlich ascites carcinoma cells was incubated with a given drug (2.5 x 10(-4) M) and then briefly cross-linked with 1% glutaraldehyde at 4 degrees C. After washing, the cells were exposed to 2% PTA (pH 7) to precipitate the drug at its binding sites. Then the samples were rapidly dehydrates in 80% ethylene glycol (4 degrees C) and embedded in the polyester, Vestopal W. This protocol provides a low denaturation, low extraction approach to tissue preparation. Control samples (without drug) demonstrated an amorphous distribution of PTA throughout the cell and no specific dense precipitates. Those cells treated with the phenothiazines (chlorpromazine or fluphenazine) or tetracycline demonstrated very discrete (4-8 nm), electron-dense drug-PTA reaction products associated with different nuclear components as well as several cytoplasmic organelles. These subcellular localizations verify the binding sites reported by the biochemical literature. In addition, several previously unresolvable binding sites are reported. The rationale and limitations of this procedure are presented. This new histochemical methodology may have broad applications in the study of drug distribution, receptors, and drug-induced pathology and toxicity that may provide new information regarding drug action and design.
Ruthenium hexammine trichloride (RHT)-mediated interaction between plasmalemmal components and pericellular matrix proteoglycans is responsible for the preservation of chondrocytic plasma membranes in situ during cartilage fixation.
Authors: Hunziker E B , Herrmann W , Schenk R K
Published: April 2011

Treatment of cartilage tissue with the cationic dye ruthenium hexammine trichloride (RHT) prior to fixation has been shown to prevent the detachment of chondrocytic plasmalemmata from the pericellular matrix and the aqueous extraction of proteoglycans during the subsequent fixation procedures. However, plasmalemmal rupture is prevented only by the simultaneous addition of RHT and the dialdehydic fixative glutaraldehyde. It is proposed that RHT forms an electrostatic cross-linkage between anionic components within the chondrocytic plasmalemma and proteoglycans of the pericellular matrix; experimental support for this hypothesis is presented. The precise nature of the plasmalemmal components with which RHT interacts is unknown. However, since their anionic properties are apparently lost following treatment with chondroitinase ABC, it seems likely that they represent chondroitin sulfate groups of membrane intercalated proteoglycans.
An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride-saponin) procedure.
Authors: Willingham M C
Published: April 2011

The use and properties of an alternative fixation procedure for ultrastructural cytochemistry is described that utilized primary fixation of cells with glutaraldehyde with considerable latitude in concentration and time of fixation. This was followed by treatment with sodium borohydride (as described by Weber, Rathke and Osborn (Proc Natl Acad Sci USA 75:1820, 1978)), which increases the accessibility of some cytoplasmic compartments to antibodies, apparently through the reduction of the Schiff bases induced by glutaraldehyde. Through the use of saponin membrane permeabilization, this primary fixation method allows good ultrastructural preservation with accessibility of most, but not all, cytoplasmic structures. Localization of tubulin, clathrin, alpha2-macroglobulin present in lysosomes, vesicular stomatitis virus (VSV) G protein, and myosin are demonstrated. This technique failed to expose the antigenic structure of actin in microfilament bundles or intranuclear antigens. This method should be useful for good preservation of ultrastructure and antibody localization of some, but not all, protein antigens in the cytoplasm of cultured cells, as well as in intact tissue. This technique is especially useful for cytosolic antigens in cultured cells.
Preparation of representative samples of subcellular fractions for electron microscopy by filtration with dextran.
Authors: Palade P , Saito A , Mitchell R D , Fleischer S
Published: April 2011

A simple method is described for obtaining a representative field of subcellular fractions for thin section electron microscopy. The prefixed sample is admixed together with high molecular weight dextran, filtered onto a Millipore filter, and embedded in polymer. Filtration serves to uniformly distribute structures of different sizes and densities. The dextran serves as a nonosmophilic spacer, increasing the space between individual structures and preventing sample compression during filtration onto Millipore filters. Sample aggregation can also readily be assessed by the procedure.
Use of low temperatures for glutathione histochemical stain.
Authors: Chieco P , Boor P J
Published: April 2011

We performed glutathione (GSH) staining at a low temperature to prevent GSH release from the section and, hence, improve morphology. Fresh frozen sections of liver, lung, kidney, heart, and stomach were incubated for GSH activity in an ice bath (2-5 degrees C) for 5-10 min. Low temperature incubation prevented GSH diffusion out of cells and minimized migration of granules into vessels and outside of tissue. Incubation at low temperature generally reduced the intensity of the stain compared to the standard method. We conclude that low temperature incubation improves GSH localization in cells, probably by regulating the rate, formation, and the size of GSH-mercury orange complexes.
Immunoelectron microscopic localization of platelet factor 4 and fibrinogen in the granules of human platelets.
Authors: Pham T D , Kaplan K L , Butler V P
Published: April 2011

To determine the storage site of platelet fibrinogen and of platelet factor 4 (PF4) in human platelets by immunoelectron microscopic techniques, washed human platelets were briefly exposed to Karnovsky's fixative and embedded in water-soluble Durcupan. Thin sections of platelets were exposed to Fab fragments of rabbit anti-human fibrinogen or of goat anti-human PF4, followed by a peroxidase conjugate of Fab fragments of antibodies to rabbit immunoglobulin (Ig) G or to goat IgG. The technique enabled preservation of the antigenic determinants of the platelet proteins, accessibility of Fab fragments to the platelet proteins, and maintenance of the ultrastructural integrity of the platelets. Using this approach, it was directly demonstrated that platelet fibrinogen and PF4 are stored in the alpha-granules of human platelets.
Immunolabeling of carbonic anhydrase isoenzyme C and glial fibrillary acidic protein in paraffin-embedded tissue sections of human brain and retina.
Authors: Kumpulainen T , Dahl D , Korhonen L K , Nyström S H
Published: April 2011

The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.
The presence of ethylenic bonds and vic-glycol groups in neuromelanin and lipofuscin in the human brain.
Authors: Barden H
Published: April 2011

Through use of oxidation and blockading reactions, chemical group precursors of aldehyde demonstrable with Schiff reaction staining were identified in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Aldehyde generation was implied by moderate staining after bleaching neuromelanin and oxidizing lipofuscin with potassium permanganate followed by oxalic acid. Confirmation of aldehyde generation was achieved when diminished staining followed a sulfite addition blockade obtained by replacing oxalic acid with metabisulfite or bisulfite as well as by condensation blockades obtained with phenylhydrazine or aniline without replacing oxalic acid. Vic-glycol precursors of aldehyde were demonstrated in both pigments when acetylation or bromination preceded permanganate-oxalic acid and staining was unequivocally diminished only after acetylation. Vic-glycols were also demonstrated in lipofuscin by diminished staining when acetylation preceded periodic acid oxidation. Ethylenic precursors of aldehyde were suggested in performic acid-bleached neuromelanin when the minimal staining that followed this peracid's generation of Schiff reaction-negative dihydroxy groups became greatly intensified following an additional oxidation with periodic acid. This additional oxidation converted the dihydroxys to Schiff reaction-positive aldehyde. Ethylenes in lipofuscin were indicated when bromination before performic acid reduced subsequent staining.
Alterations in membrane sugars during epidermal differentiation: visualization with lectins and role of glycosidases.
Authors: Nemanic M K , Whitehead J S , Elias P M
Published: April 2011

Differentiation in keratinizing epithelia involves the orderly transformation of basal germinal cells into an exterior cornified layer. We have employed rhodamine-conjugated lectins to visualize distinctive changes in the localization of keratinocyte membrane glycoconjugates during epidermal differentiation. The dermis, basement membrane, and epidermal cell membranes stained positively for mannose, alpha- and beta-galactose, N-acetyl-glucosamine, and sialic acid. In contrast, only the viable epidermis demonstrated N-acetyl-galactosamine, while alpha-L-fucose staining was limited to the upper stratum spinosum and stratum granulosum. Neuraminidase treatment extended the binding of certain lectins, e.g., peanut agglutinin, to regions of the skin that otherwise did not label. Whereas the granular cell membranes displayed the largest number of carbohydrates, these sugars could no longer be visualized after granular cells differentiated into the stratum corneum. Loss of lectin staining may be attributable to the presence of a family of sugar-specific glycosidases that we obtained from granular and cornified cell cytosol fractions. Finally, as further support for sugar deletion during cornification, we found that glycosphingolipids are hydrolyzed to ceramides coincident with both loss of lectin staining and the emergence of glycosidase activity. These results suggest: 1) that carbohydrates on keratinocyte cell membranes can be used as markers of epidermal differentiation, and 2) that removal of cell surface sugars during cornification may be due to the action of specific glycosidases in the outer epidermis.
Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods.
Authors: Nagle R B , Clark V A , McDaniel K M , Davis J R
Published: April 2011

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.