bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.
Authors: Mesulam M M , Rosene D L
Published: April 2011

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.
The labeled antigen method of immunoenzymatic staining.
Authors: Mason D Y , Sammons R E
Published: April 2011

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.
Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.
Authors: Baskin D G , Erlandsen S L , Parsons J A
Published: April 2011

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.
Catalase in skeletal muscle fibers.
Authors: Christie K N , Stoward P J
Published: April 2011

Catalase has been localized immunocytochemically with anti-bovine catalase in long thin filament structures in aerobic type I fibers in the skeletal muscles of normal and genetically dystrophic hamsters. The filaments range in length from 1 to 60 micron, are orientated regularly along the long axis of the fibers, and also seem to surround and project from muscle nuclei. The enzyme thus appears to be more prominent in the sarcoplasmic reticulum than in peroxisomes, and in this situation is suitably placed for destroying toxic hydrogen peroxide which may be continously generated in aerobic fibers.
Immunohistochemical characterization of monolayer cell cultures of embryonic chicken pancreas and measurement of somatostatin release.
Authors: Barden N , Dubé D , Côté J P , Leclerc R , Pelletier G
Published: April 2011

Monolayer cell cultures of embryonic chicken pancreas contain functionally active insulin, glucagon and somatostatin-containing cells as evidenced by immunohistochemical and radioimmunoassay techniques. Hormone release is in relation to the number of each cell type present and responds to known specific secretory stimuli. The relatively high numbers of D-cells and amounts of immunoreactive somatostatin released by this preparation makes this system a suitable model for studies of somatostatin function and secretion.
Immunocytochemical localization of hepatic legandin and Z protein utilizing frozen sections for light and electron microscopy.
Authors: Capron F , Coltoff-Schiller B , Johnson A B , Fleischner G M , Goldfischer S
Published: April 2011

Ligandin (glutathione-s-transferase) and Z protein are soluble hepatocellular proteins that are involved in the transfer of organic ions, including bilirubin and some hormones and carcinogens from the plasma to the liver. The intracellular distribution of ligandin and Z protein was studied by applying the peroxidase-antiperoxidase procedure of L. A. Sternberger (Immunocytochemistry, Prentice Hall Inc., 1974) to paraffin sections and free-floating 10-micrometers frozen sections that were processed for both light and electron microscopy. Ligandin and Z protein were localized to the cytosol of hepatocytes in association with smooth endoplasmic reticulum (SER), but no reaction product was present between cisternae of rough endoplasmic reticulum. Penetration of reagents was enhanced in 10-micrometers frozen sections and the preservation of subcellular structures was equivalent to thicker, unfrozen sections.
Morphometrical analysis of an ultrahistochemical demonstration of nonspecific esterases in hepatocytes of mice after fructose overload.
Authors: Böcking A , Riede U N
Published: April 2011

Changes in ultrastructural distribution patterns of nonspecific esterases (E.C. 3.1.19) are described quantitatively by means of morphometry. Esterases were demonstrated with O-acetyl-8-hydroxyquinoline (Q-O-2) and S-acetyl-8-mercaptoquinoline in livers of normally and exclusively fructose-fed mice. Conditions are discussed, under which the quantification of the ultrastructural products of enzyme histochemical reactions may be possible. Smooth endoplasmic reticulum and rough endoplasmic reticulum exhibit no alteration in enzyme distribution with both substrates since the enzyme-occupied proportion of each compartment remains the same despite an overall decrease of both compartments. Likewise an increase of O-acetyl-8-hydroxyquinoline-esterase at fat droplets corresponds to the increase in total surface of the fat. S-acetyl-8-mercaptoquinoline-positive fat surface however reveals an increase far beyond that of the total fat surface. The results support the hypothesis that a variety of esterases with different substrate spectra are present at the subcellular level in different cell compartments.
Immunoferritin location of carbamoyl phosphate synthetase in rat liver.
Authors: Knecht E , Hernández J , Wallace R , Grisolía S
Published: April 2011

Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.
Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method.
Authors: Cournil I , Leblond C P , Pomponio J , Hand A R , Sederlof L , Martin G R
Published: April 2011

Frozen sections of the growing end of the rat incisor tooth were exposed to antisera or affinity prepared antibodies against partially purified type I, II, or IV procollagen in the hope of detecting the location of the corresponding antigens by the peroxidase-anti-peroxidase technique. The distribution of immunostaining was similar with antisera as with purified antibodies of a given type, but differed for each type; that is, predentin, odontoblasts, pulp and periodontal tissue were the sites of type I; blood vessel walls, pulp and periodontal tissue, of type III; and basement membranes, of type IV antigenicity. It was demonstrated, at least in cases of type I and III, that immunostaining detected the corresponding procollagens and related substances, but not the corresponding collagens. The interpretation of these observations is that: 1) odontoblasts elaborate procollagen I for release to predentin and subsequent transformation to dentinal collagen I; 2) pulp and periodontal cells produce procollagens I and III which presumably become collagens I and III respectively, while the adventitial cells of blood vessels give rise to collagen III; and 3) procollagen IV is associated with basement membranes and, occasionally, adjacent cells.
Diagnostic applications of an immunoperoxidase method in hematopathology.
Authors: Banks P M
Published: April 2011

The diagnostic usefulness and reliability, in particular in relation to methodologic problems and limitations, of an immunoperoxidase method are discussed, based upon an initial 3-yr experience with more than 400 cases. It has been found necessary to employ certain control preparations in each study case in order to avoid interpretational errors which can derive from variation in distribution of plasma proteins, in preservation of immunoreactivity with different fixatives, and in nonspecific binding of antiserum. The method is suitable for detection of immunoglobulin components only among lymphoid cells corresponding to late stages in the (B-cell) sequence of transformation. One case has been identified in which the results of the method are inconsistent with additional data, that of large cell neoplasm of apparent epithelial nature with a monotypic immunoglobulin staining pattern. Cytoplasmic lysozyme was present in a variety of different disease processes, including adenocarcinoma and granulocytic, monocytic, and histiocytic proliferations.
Enzymatic tracers in the study of vascular permeability.
Authors: Simionescu N
Published: April 2011

Elucidation of the ultrastructural basis of vascular permeability was aided by the development of cytochemical techniques for visualizing the distribution, within the vessel wall, of intravenously injected peroxidatic enzymes of varying molecular size. Tracer enzymes available range from 10 A (hemeoctapeptide) to 52 A (catalase) effective molecular radius. The use of enzymatic probe molecules assumes a thorough characterization of: (a) the molecular charge (isoelectric point of the native enzyme, and when feasible, its polyanionic and polycationic derivatives; (b) effective molecular radius (ae); (c) peroxidase activity (to detect by spectrophotometry of DAB-oxidizing activity, the optimal pH, temperature, and enzyme concentration to be employed in the cytochemical procedure). Molecular shape and state of dispersion of the enzymatic probes should be determined by gel chromatography and spectrophotometry of both the tracer solution and aliquots of blood plasma collected after i.v. injection of the tracer. Conditions required for the probe administration include: (a) the investigation of potential side effects (tests for toxicity and vascular leakage) and (b) estimation of the tracer volume and concentration which does not affect significantly the blood volume and osmotic pressure. Determination in vitro of the crosslinking of tracer molecules induced by the aldehyde fixative to be employed, also gives an indication on potential diffusion artifacts. Based on the information thus obtained, the design of the cytochemical procedure should also take into account the possible use of methods for enhancing the peroxidatic reaction product: nitrogenous ligands (imidazole, diaminopyrimidine, histidine) or polyphenolic mordants (galloylglucoses). The usefulness of peroxidatic tracers in the investigation of vascular permeability is exemplified by some results obtained on the microvascular endothelium in vivo (trasncytosis, intercellular pathway, etc.), and on endothelial cells isolated from heart microvasculature.
The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.
Authors: De Bruyn P P
Published: April 2011

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).
The histochemistry of colonic mucins.
Authors: Culling C F , Reid P E
Published: April 2011

This presentation could perhaps better be titled the histochemistry of colonic mucins, as based upon PAS reactivity, since it mainly concerns development of methods for the identification of the various patterns of o-acylation of sialic acid as measured by alteration in PAS staining.
Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.
Authors: Al-Ali S Y , Robinson N
Published: April 2011

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.
Ultrastructural characteristics of freeze-dried smooth muscle.
Authors: McGuffee L J , Little S A , Hurwitz L
Published: April 2011

The rapid freezing and vacuum dehydration of tissue has been employed to study the intracellular distribution of diffusible substances at the electron microscopic level. Hower, the ultrastructural detail of freeze-dried tissue is difficult to retain. Consequently, the ultrastructural preservation of freeze-dried smooth muscle has not been sufficient to permit satisfactorily definition of intracellular organelles. Therefore, determinations of the intracellular distribution of soluble ions have not been achieved in freeze-dried smooth muscle. In this study a freeze drying method is presented which provides more satisfactory definition of intracellular organelles than has been available in the past. Using this merens have been preserved. When these muscles are compared to convention preparer of surface vesicles that are observed, and the mitochondria are extremely electron opaque in the freeze-dried tissue. The smooth muscles which have been examined do not have the inherent contrast of other types of tissue nor do they contain the different types of mitochondria that have been observed in nonmuscle tissue.
Cytochemical studies on the relation of nucleoside triphosphatase activity to ribonucleoproteins in isolated rat liver nuclei.
Authors: Vorbrodt A , Maul G G
Published: April 2011

Cytochemical tests for nucleosidetriphosphatase (NTPase) and Bernhard's preferential staining for ribonucleoproteins (RNP) were applied to isolated rat liver nuclei. The strongest and most easily reproducible positive reaction for NTPase was detected at pH 7.7 with ATP and GTP. This reaction was activated by Mg2+ and Ca2+ and inhibited by Be2+, Zn2+, quercetin, and ribonuclease. The major sites of enzyme reaction were intranuclear RNA-containing structures. Incubation of nuclei in ATP-stimulated RNA-release medium eliminated a considerable part of the material showing both NTPase reaction and staining for RNP; the perichromatin granules disappeared, while interchromatin granules remained. NTPase activity in the nuclear envelope seems to be associated with the annular part of nuclear pore complexes (permanent component) and with RNP particles translocated through nuclear pores or attached to the surface of nuclei (transitional component). From a morphological point of view, these observations support previous biochemical data suggesting the existence of a connection between NTPase activity and the translocation of RNP particles through the nuclear envelope.
Histiocyte X positivity for nonspecific esterase.
Authors: Jaubert F , Barbey S , Nogues C , Monnet J P , Grun M , Nezelof C
Published: April 2011

The cell membrane and X bodies of histiocytes X from two cases of eosinophilic granuloma were stained by nonspecific esterase. The results show that histiocytes X possess a cell membrane exoenzyme similar to that of other histiocytes, such as alveolar macrophages, but the role of the X body as related to enzyme activity remains an unknown.
Trypsin digestion in immunoperoxidase staining.
Authors: Hautzer N W , Wittkuhn J F , Mccaughey W T
Published: April 2011

The effect of trypsin digestion on the immunoperoxidase-PAP staining of paraffin sections from normal and abnormal tissue was studied. Trypsin treatment reduced background staining considerably, but enhanced specific staining. Results were, therefore, much easier to read. Possible reasons for this result are discussed.
Ultrastructural localization of elastase-like enzymes in human neutrophils.
Authors: Clark J M , Aiken B M , Vaughan D W , Kagan H M
Published: April 2011

The thiol ester, N-t-butyloxycarbonyl-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) has been synthesized and applied as an ultrastructural cytochemical substrate for leukocytic elastase-like enzymes. Incubation of fixed human neutrophils with Boc-Ala-SNp in the presence of gold ions generates electron-dense deposits of gold p-nitrothiophenolate in the nuclear membrane, endoplasmic reticulum, Golgi complex, mitochondria, and granules. Deposition of product is inhibited by pretreatment of cells with general and specific chemical inactivators of neutrophil elastase. This substrate appears to have significant potential as a probe for the ultrastructural localization of elastase-like activity.
Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells.
Authors: Oliver C
Published: April 2011

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.
Preparation and characterization of peroxidase: antiperoxidase-Fab complex.
Authors: Slemmon J R , Salvaterra P M , Saito K
Published: April 2011

The preparation and characterization of a horseradish peroxidase-rabbit antiperoxidase Fab immunocomplex (HRP-Fab2) useful for immunocytochemical localization of primary tissue-bound rabbit antibody are described. Antisera with titer to horseradish peroxidase (HRP) were raised in rabbits. Anti-HRP-Fab fragments were prepared by controlled mercuripapain digestion of the purified rabbit IgG. The complex was formed during incubation of Fab fragments with HRP, and fractions containing HRP activity that were precipitable by goat anti-rabbit IgG serum were isolated by gel filtration. The major isolated complex had a molecular weight of approximately 150,000 daltons and migrated as a single band on cellulose acetate electrophoresis. Polyacrylamide gel electrophoresis in SDS indicated the major polypeptide components of the complex were HRP and Fab. RZ (absorbance at 403 nm/275 nm) determination indicated a molar ratio of 2 Fab:1 HRP. The complex was stable for at least 1 year at -20 degrees C and was used successfully in a number of immunocytochemical procedures.
Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity to the intermediate saccules of the Golgi apparatus in rat incisor ameloblasts.
Authors: Smith C E
Published: April 2011

Cytochemical evidence for the existence of a Golgi-associated phosphatase activity that hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) at acid pH in rat incisor ameloblasts was obtained by incubating sections from glutaraldehyde-fixed teeth in a medium containing NADP as substrate and lead ions as capture agent. Following incubation for 1 hr at 37 degrees C and pH 5.0, the Golgi saccules situated between those at the cis (immature) and trans (mature) faces of the ameloblast Golgi apparatus were marked by reaction product with the heaviest deposit in the middle saccule. Reaction product was otherwise seen in trace amounts only over some elements of the GERL system as well as a few lysosomal dense bodies and immature secretory granules. Control experiments established that the selective staining of intermediate Golgi saccules at pH 5.0 could only be duplicated by using substrates that resembled the complete NADP molecule, and not just the portion containing the adenosine 2'-monophosphate group. As well, no deposits of reaction product were seen within the Golgi saccules of ameloblasts incubated at pH 5.0 with nictoinamide adenine dinucleotide (NAD) as the substrate or that were incubated at pH 7.2 or pH 9.0 with NADP as the substrate. It was concluded that a specific, acid-NADPase activity is present in the intermediate Golgi saccules of secretory ameloblasts. Preliminary observations on other cells suggest that the localization of NADPase activity to Golgi saccules may constitute a general phenomenon.
Cytochemical differentiation of the Golgi apparatus from GERL.
Authors: Hand A R
Published: April 2011

GERL exhibits a number of structural properties, such as its location at the trans face of the Golgi apparatus, thick limiting membranes with occasional coated regions, and close relationship to the ER, which are similar in all cells wehre it has been identified. Acid hydrolase activity and the formation of lysosomes, also considered characteristic of GERL, have been shown to be less consistent properties of GERL in some cells. Finally, GERL and the Golgi saccules of certain cells undergo significant changes in their cytochemical and structural properties in response to specific alterations in cellular activity. These variations are important for at least two reasons: first, they indicate the care required in differentiating GERL from the Golgi saccules based on limited cytochemical observations; second, and most important, they may yield information regarding the biogenetic and functional relationships between the ER, Golgi saccules, and GERL, such as the origin of GERL in various cells and the role of each organelle in the processing of lysosomal and secretory proteins.
Evaluating optical filter efficiency in a flow cytometer.
Authors: Loken M R
Published: April 2011

A simple yet effective method of testing optical filters under normal operating conditions on a Fluorescence Activated Cell Sorter is described. The spectral characteristics of three filters are compared to demonstrate the methods of determining which filter is the best in a particular situation. These tests include determining the efficiency of transmitting fluorescent light, determining the efficiency of removing scattered light, and detecting fluorescence originating from the filter itself.
High voltage electron microscopy of membrane interactions in wheat.
Authors: Marty F
Published: April 2011

Structural interactions between intracellular membranes in root cells of Triticum sativum L. have been investigated with the 3 MeV electron microscope in Toulouse after selective labeling of the membranes with either zinc iodide-osmium tetroxide or phosphotungstate at low pH. Direct membrane continuities between endoplasmic reticulum, Golgi apparatus, and biogenetically derived compartments of the exoplasmic space are more extensive than commonly suggested from conventional electron microscopy. The results are discussed with reference to current concepts suggesting a membrane flow from the endoplasmic reticulum to the terminal components, plasmalemma, and vacuoles.
A small tissue sample, rapid freeze-drying method for monoamine histochemistry.
Authors: Carrasco N J , Carrasco V N , Goulian D
Published: April 2011

A simple technique, and an inexpensive, easily assembled freeze-drying apparatus is described. This system rapidly dries small tissue samples to give good reproducible results in preparation for the histochemical visualization of monoamines, by utilization of liquid nitrogen and dry ice sublimation under vacuum.
Cytochemical localization of ouabain-sensitive (K+)-dependent p-nitrophenyl phosphatase (transport ATPase) in human blood platelets.
Authors: Cutler L S , Feinstein M B , Christian C P
Published: April 2011

The ultrastructural cytochemical localization of a potassium-dependent oubain-sensitive nitrophenyl phosphatase (transport ATPase) activity in human blood platelets is described. This potassium-dependent nitrophenyl phosphatase activity was not affected by 5 mM levamisole, indicating that the reaction product identified was not due to nonspecific alkaline phosphatase activity. The K+-dependent nitrophenyl phosphatase was strictly localized to the platelet plasma membrane, while the open canalicular system and dense tubular system were devoid of reaction product. In contrast, (Ca2+,Mg2+)-activated ATPase activity was predominantly localized in the open canalicular system and dense tubular system with very little cytochemical activity expressed at the plasma membrane. These data demonstrate a relative segregation of these enzymes into unique membrane compartments of the human platelet. Such data may be useful with regard to identification of purified membrane fractions from platelets and may be significant with regard to the understanding of the function(s) of the different membrane compartments of the human platelet.
Specific immunohistochemical localization of type III collagen in porcine periodontal tissues using the peroxidase-antiperoxidase method.
Authors: Wang H M , Nanda V , Rao L G , Melcher A H , Heersche J N , Sodek J
Published: April 2011

Specific antibodies to porcine gingival type III collagen were raised in sheep. After purification on collagen affinity columns the antibodies were used for immunohistochemical localization of type III collagen in porcine periodontal and dental tissues employing the peroxidase-antiperoxidase (PAP) procedure. The extent of staining of the periodontal tissues was found to approximate the amount of type III collagen measured biochemically. A fairly uniform distribution of type III collagen was observed in the periodontal ligament and gingiva with more intense staining often being associated with blood vessels. A regular pattern of weakly staining fibers could be demonstrated throughout the cementum and in parts of the alveolar bone tissue. In addition, occasional sites in the cementum having a different morphological appearance from the rest of the cementum exhibited bundles of positively stained fibers. Although the bone tissue was essentially unstained, fibers in the endosteal spaces stained strongly. Sharpey's fibers passing from the soft connective tissues into alveolar bone and cementum also stained strongly. Three distinct arrangements of collagen fibers stained by the type III collagen antibodies could be identified: first, a reticular pattern, which was seen at the junction of the gingival epithelium and connective tissue, and in the endosteal spaces and dental pulp; second, a more diffuse pattern of fibers intermingled with type I collagen in the soft connective tissues; and third, a coating of some Sharpey's fibers, having a core believed to be type I collagen, and of fibers in the cementum inclusions.
Immunohistochemical localization of hexose 6-phosphate dehydrogenase in various organs of the rat.
Authors: Tanahashi K , Hori S H
Published: April 2011

Distribution of hexose 6-phosphate dehydrogenase in various organs of the rat has been studied by a peroxidase-labeled antibody method in order to find some clue to elucidating the, as yet unclear, function of this enzyme. As a result, the following cells were found to contain this enzyme in relative abundance: hepatic parenchymal cells, ovarian lutein and theca interna cells, testicular interstitial cells, striated ducts and serous tubular portions of the submandibular gland, plasma cells and the P3 segment of proximal convolutions, and collecting tubules of the cortex and inner medulla of the kidney. Although the role of this enzyme in salivary glands and in plasma cells is unclear at present, the results obtained with steroidogenic cells, liver cells, and renal tubules appear to suggest the possibility that this enzyme might be involved in drug and steroid metabolism.
Use of the protein A-gold technique for the morphological study of vascular permeability.
Authors: Bendayan M
Published: April 2011

The protein A-gold immunocytochemical technique has been applied for the ultrastructural study of vascular permeability. A capillary preparation was perfused for 30 min with a buffer solution containing albumin. After fixation and embedding, the albumin molecules were revealed on the tissue sections using anti-albumin antiserum and the protein A-gold complex. A specific labeling was obtained over the capillary lumen, the vesicular structures of the endothelial cells, and the intercapillary space. These results allowed us to conclude that the protein A-gold technique is suitable and of general applicability for the morphological study of vascular permeability.
Cytochemical localization of a "basic" ATPase to canine myocardial surface membrane.
Authors: Malouf N N , Meissner G
Published: April 2011

Enzymatic properties of a canine cardiac muscle microsomal fraction were determined to localize in situ a "basic," divalent cation dependent adenosine triphosphatase (ATPase) by ultrastructural cytochemistry. The microsomal fraction had a buoyant density of 1.08--1.13 (20--30% [w/w] sucrose) and hydrolyzed adenosine triphosphate in the presence of Mg2+, Ca2+, Mn2+, or Co2+, but not in that of Sr2+ or Ni2+, under conditions that inhibited interfering (Na+ + K+)-ATPase and sarcoplasmic reticulum Ca2+-ATPase activities. "Basic" ATPase was localized in paraformaldehyde-fixed tissue in a medium containing Mg2+ or a high Ca2+ concentration (4 mM). A free Pb2+ concentration of less than 1 microM was used to capture enzymatically released phosphate anions. Electron-dense lead precipitates were present at the plasmalemma, T-system, and intercalated disc membranes with the exception of the nexus. These studies suggest that "basic" ATPase activity is associated with surface membrane structures of canine cardiac muscle.
Localization of kallikrein in submandibular gland of cat, guinea pig, dog, and man by the immunoperoxidase method.
Authors: Schachter M , Peret M W , Moriwaki C , Rodrigues J A
Published: April 2011

The sensitive immunoperoxidase method was used to localize kallikrein in the submandibular gland of the cat, guinea pig, dog, and man. In every instance, kallikrein was localized in the apical region of duct cells, a location suggesting its secretion into the duct system. There was no evidence of the enzyme in other cells or interstitial tissue in the gland. The major source of submandibular gland kallikrein, therefore, must be of ductal origin. The classification of kallikreins with the widely distributed group of serine proteases is discussed, as is their possible significance in regulating physiological processes by specific and limited proteolysis.
Differential expression of surface monosialoganglioside GM1 in various hemic cell lines of normal human bone marrow. A quantitative immunocytochemical study using the cholera toxin-gold-labeled anti-cholera toxin procedure.
Authors: Ackerman G A , Wolken K W , Gelder F B
Published: April 2011

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anti-cholera toxin ultrastructural immunocytochemical procedure has been used for the localization of GM1 monosialogangliosides on the surface of human bone marrow cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various types of marrow cells, although minor quantitative differences were noted in surface labeling densities between subjects. Surface labeling was nonuniformly distributed along the cell membrane of the marrow cells and label clusters or domains were commonly noted. Data analysis indicated that CT labeling was related to cell type, to cell lineage, and to the stage of maturation. Mature neutrophils were the most reactive of the marrow cells and the CT labeling of this cell series increased stepwise from the promyelocyte stage to the segmented neutrophil. A similar pattern occurred during eosinophil maturation and the maturation of the monocyte. A different labeling pattern was found during the differentiation of the erythrocytic cell series with low labeling of proerythroblasts increasing modestly to the early normoblast stage and then decreasing during the final phase of maturation. Exposure to neuraminidase prior to the immunocytochemical sequence induced a major increase in surface CT labeling of the various types of marrow cells, as was particularly evident for the platelet, promyelocyte, myelocyte, monocyte, promonocyte, and erythrocyte cell groups. The data indicated that the number of cryptic GM1 and/or higher gangliosides exposed by neuraminidase in the cell membrane varied during cell differentiation and was directly related to specific cell types. Exogenous GM1 also was demonstrated to be incorporated into the surface of the bone marrow cells in a differential manner and the extent of incorporation was found to be related to specific cell types and to their stage of maturation.
A rapid, routine technique for the X-ray microanalysis of microincinerated cryosections: an SEM study of inorganic deposits in tissues of the marine gastropod Littorina littorea (L.).
Authors: Mason A Z , Nott J A
Published: April 2011

A procedure is described that prepares chemically untreated biological sections for X-ray microanalysis in the scanning electron microscope (SEM). The method aims to retain and localize labile components in tissue sections by a procedure that is both rapid and routine. Large quantities of fresh tissue can be processed for analysis within a single day. Thick cryosections are cut with a steel knife in a conventional cryostat, freeze-dried, and then ashed by either low or high temperature incineration procedures. Controlled microincineration attenuates the organic matrix to reveal sufficient surface relief for effective SEM of some cytological structure and microanalysis of the residual inorganic components. The detectability of various elements is enhanced because the relative concentrations in the residues are increased and the level of nonspecific background in the X-ray spectra is reduced. The technique is applied to different tissues from the visceral complex of the marine prosobranch Littorina littorea. In animals exposed to elevated levels of zinc it can be demonstrated tht the metal is localized both as an insoluble form in granules and as a labile form within the cytoplasm. Other metals, including magnesium, potassium, calcium, manganese, and iron, have been identified and localized. The effectiveness of this technique for retaining labile elements is compared, in outline, with that of conventional fixation procedures.
The presence of mast cell granules in rat parotid secretory granule preparations.
Authors: Robinovitch M R , Lagunoff D , Iversen J M
Published: April 2011

Rat parotid secretory granule preparations contain, in addition to the acinar secretory granules, a second type of granule. Whereas the acinar granules lyse under hypotonic conditions, this second type of granule does not, thus providing a means for obtaining a fraction sufficiently enriched in these granules to allow for their characterization. In the present study, these granules are shown to possess demonstrable chymotrypsin-like enzyme activity. In the intact rat parotid, such activity is shown by histochemical methods to be present in the numerous mast cells residing in the connective tissue stroma, but no such activity exists in any of the parenchymal cells. On the basis of their electron microscopic appearance, enzyme activity, and physical characteristics it is concluded that the second type of granule present in rat parotid secretory granule preparations originates from stromal mast cells rather than from parenchymal cells.
An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina.
Authors: Fry K R , Spira A W
Published: April 2011

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.
Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol.
Authors: Robinson J M , Karnovsky M J
Published: April 2011

Distribution of the filipin-cholesterol complexes has been studied by freeze-fractures techniques. While filipin-cholesterol complexes are formed in fixed as well as unfixed cells, the distribution of the complexes varies with fixation. Furthermore, filipin induces dramatic rearrangement of intramembrane particles in unfixed, but not in fixed, cells. Examination of thin sections of filipin-treated cells shows that filipin alone induces substantial cellular damage, while filipin treatment simultaneous to fixation or following fixation does not cause noticeable damage. For filipin to be an effective cytochemical probe of membrane structure, cellular damage must be minimized; this is best achieved by treating cells with filipin following glutaraldehyde fixation.
Quantitative immunocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell.
Authors: Bendayan M , Roth J , Perrelet A , Orci L
Published: April 2011

The recently developed protein A-gold technique for the detection of intracellular antigenic sites on thin sections was utilized to localize nine different secretory proteins in the rat exocrine pancreas. Amylase, chymotrypsinogen, trypsinogen, lipase, elastase, carboxypeptidases A and B, RNase and DNase, were detected at the level of the rough endoplasmic reticulum, the Golgi area, and the zymogen granules of the acinar cells, as well as in the acinar lumen. A quantitative evaluation of the labeling showed that its intensity was not identical for all enzymes studied nor in all cellular compartments analyzed. An increasing gradient of the labeling from the rough endoplasmic reticulum to the Golgi and to the zymogen granules was found for amylase, carboxypeptidases A and B, chymotrypsinogen, trypsinogen, and RNase, while a comparable low degree of labeling in the Golgi apparatus and in the zymogen granules was observed for DNase, lipase, and elastase. These results suggest that the nine enzymes are processed through the same intracellular compartments, but that they may be concentrated to different degrees in the zymogen granules before being released in the acinar lumen.
A rapid method for accurate DNA measurements in single cells in situ using a simple microfluorimeter and Hoechst 33258 as a quantitative fluorochrome.
Authors: Cowell J K , Franks L M
Published: April 2011

A method is described for the rapid, accurate measurement of single cells in situ using microfluorimetry. This method involves a shutter system, which allows irradiation of single cells for fractions of a second and a peak fluorescence intensity recording device. In this way errors due to fluorochrome fading are almost eliminated and standard deviations of less than 5% are obtained. Hoechst 33258 has been used as a quantitative fluorochrome. Optimum fixation and staining conditions on glass and plastic tissue culture vessels are described.
Immunohistochemical localization of cyclic GMP in goldfish retina.
Authors: Ortez R A , Sikes R W , Sperling H G
Published: April 2011

The immunocytochemical procedure for cyclic GMP (cGMP) localization has been modified by the development of: 1) an anti-cGMP antibody depleted control serum and 2) a tissue preadsorption procedure that greatly reduces nonspecific fluorescence. cGMP remaining associated with tissue sections following prolonged exposure to aqueous buffer was determined, by extraction and measurement by radioimmunoassay, to be approximately 38% of the initial cGMP content. Application of this improved immunohistochemical procedure to goldfish retinas reveals the highest concentrations of cGMP to be associated with the cone inner segments and nuclei of the photoreceptors. This retinal distribution of cGMP is in good agreement with that independently determined by microdissection studies. The validity of this procedure is therefore strengthened and cautions to its application and interpretation are outlined.
Determination of immunoreactive melatonin in the colon of the rat by immunocytochemistry.
Authors: Holloway W R , Grota L J , Brown G M
Published: April 2011

Melatonin immunofluorescence in the rat colon was measured with a cadmium sulfide photometer in conjunction with an immunohistological double antibody procedure. The method proved to be very reliable with 5-10% intra- and interassay reliabilities. In addition, manipulation of the primary antibody concentration, the incubation time, and the nature of the first antibody produced predicted changes in fluorescence intensity. Treatment with p-chlorophenylalanine led to a marked reduction of melatonin-specific fluorescence.
An immunofluorescence procedure for the tissue localization of glucosephosphate isomerase.
Authors: Gearhart J , Oster-Granite M L
Published: April 2011

Anti-glucosephosphate isomerase antibodies have been used to reveal the presence of the glycolytic and gluconeogenic enzyme, glucosephosphate isomerase (GPI) in mouse tissues by an indirect immunofluorescence technique. The enzyme was localized in 6-micron sections from tissues embedded in paraffin. GPI was observed in the sarcoplasm of skeletal muscle, all hepatocytes of the liver, and in the granulosa cells and oocytes of growing ovarian follicles. The immunofluorescence procedure circumvents the problems associated with the conventional localization of GPI by histochemistry and appears to be a more sensitive procedure for the detection of low concentrations of GPI, as demonstrated by the staining of the ovarian follicles.
Immunohistochemical demonstration of Fc receptors of human lymph node cells using peroxidase-antiperoxidase (IgG).
Authors: Itoh G
Published: April 2011

Human lymph node cells, prepared from regional lymph nodes excised from four patients with gastric cancer, were incubated with peroxidase-antiperoxidase (IgG) (PAPIgG). After being washed, they were reacted with diaminobenzidine tetrahydrochloride in the presence of H2O2. Light microscopic examination revealed that a certain proportion of lymph node cells (18.2-32.2%) were labeled on their cell surface with brown-colored reaction products and that the labeled cells were composed of small lymphocytes. Electron microscopic examination demonstrated electron-dense irregular-shaped aggregates of reaction products on the cell surface of lymphocytes. Characterization experiments confirmed that the immune complexes of PAPIgG bound specifically with Fc receptors. PAPIgG, therefore, can be used as a specific indicator for Fc receptor of human lymph node cells.