bluuebunny/crossref_metadata_2025 huggingface dataset:
Scintillation fluid shortens exposure times in autoradiography.
Authors: Stanulis B M , Sheldon S , Grove G L , Cristofalo V J
Published: April 2011

Premixed, commercially available scintillation fluids were used to reduce exposure time of tritium (3H) and iodine-125 (125I)-labeled whole cells, and of 3H-labeled Giemsa-banded chromosome preparations. Emulsion-coated slides were dipped into scintillator for no longer than 2 min and exposed in the dark at 4 degrees C. Maximal values for percentage of human diploid cell (WI-38) nuclei labeled with 3H-thymidine of moderate specific activity were obtained in 12 hr. Without scintillator the exposure time was 4 days. Exposure time for cells labeled with 125I-serum was reduced from over 90 days to 14 days. The shortened exposure time for banded chromosomes permitted successful prestaining with Giemsa, a sequence that is not possible without scintillator.
Adenylate cyclase activity in the parotid gland of the mouse after isoproterenol stimulation.
Authors: Revis N W , Durham J P
Published: April 2011

Previous studies have described a decrease in the activity of adenylate cyclase in the parotid gland of isoproterenol-treated rats. In the present studies, a similar decrease was observed in mice treated with isoproterenol. Studies on the subcellular distribution of adenylate cyclase after isoproterenol stimulation of the parotid gland showed that enzyme activity was increased in the lysosomal fraction and decreased in the cellular membrane fractions. Cytochemical studies on the localization of adenylate cyclase in stimulated gland showed an increase in vesicles which contained enzyme activity and a decrease in activity at the luminal and plasma membranes. It is suggested, based on the present findings and results reported by other investigators, that after isoproterenol stimulation of the parotid gland, adenylate cyclase (along with excess membrane) is degraded by lysosomes. If this suggestion is true, then the observed decrease in adenylate cyclase would have a molecular explanation.
Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology.
Authors: Walton J
Published: April 2011

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.
Horseradish peroxidase as a tracer for capillary permeability studies.
Authors: Weihe E , Nimmrich H , Metz J , Forssmann W G
Published: April 2011

The permeability of capillaries was investigated utilizing an in vivo injection of horseradish peroxidase (HRP) and an in situ perfusion of a balanced salt solution containing HRP and lanthanum chloride. In the continuous capillaries of heart and muscle, HRP diffuses mainly through intercellular junctions, while in testicular capillaries, the transport is via micropinocytotic vesicles. The diffusion and micropinocytotic transport of HRP was demonstrated in both directions, i.e. from the capillary lumen to the interstitium and vice versa. Lanthanum can be used as a bidirectional inhibitor of micropinocytosis. The transport of HRP is then almost completely hindered in testicular capillaries. In heart muscle, the effect on HRP transport is not significant, due to second transport pathway, i.e. intercellular cleft passage.
The diffuse neuroendocrine system. Studies of this newly discovered controlling system in health and disease.
Authors: Polak J M , Bloom S R
Published: April 2011

Numerous peptides (neuropeptides) have been recently found to be present in both the nervous and endocrine systems composing what is now known as the diffuse neuroendocrine system. Two immunological methods, radioimmunoassay and immunocytochemistry, have been used here in combination to study their distribution and cellular localization. A number of these neuropeptides have recently been found to be abnormal in disease state, thus providing further information as to their role in normal and pathological conditions.
Nonspecific light loss and intrinsic DNA variation problems associated with feulgen DNA cytophotometry.
Authors: Miksche J P , Dhillon S S , Berlyn G P , Landauer K J
Published: April 2011

Nonspecific light loss by the cell-wall-plus-cytoplasm (CWC) can cause a 50% increase in Feulgen absorption units in peanut root-tip nuclei as determined by scanning at 450 nm, whereas this phenomenon is not evident with chicken erythrocytes. A two wavelength scanning method of subtracting nonspecific 450 nm absorption from 550 nm Feulgen absorption values eliminated the nonspecific light loss in CWC, However, the two wavelength scanning method is time consuming and somewhat impractical with a regular scanning microdensitometer such as Vickers M85. Elimination of the problem of nonspecific light loss is suggested by careful determination of background setting with the spot position close to the nucleus in CWC. The accuracy of the CWC background setting method was further tested by comparison with subtraction method. The use of plant nucleis as an internal standard in plant DNA measurements was also evaluated. Significant variation among the replicate slides due to the variation in pine nuclear DNA amounts was observed and plant nuclei generally are not reliable internal standards. Mature chicken erythrocytes are recommended as an internal standard because the cell type and metabolic state is known.
Applications of microcomputer technology to cytophotometry.
Authors: Rasch E M , Rasch R W
Published: April 2011

Lack of appropriate software support for microprocessor program development has previously limited the applications of such technology in the field of microspectrophotometry. This paper describes our use of a Vickers M86 integrating microdensitometer coupled through a custom-designed interface circuit to a Processor Technology SOL III microcomputer. W e have developed a series of interactive, user-oriented programs for DNA-Feulgen cytophotometry with this instrument to allow automatic storage of data in files on floppy disks and instant retrieval of sets of measurements for statistical processing. The same data files can also be used to generate graphic displays in the form of bar histograms or plots of linear regressions on a video monitor and to produce hardcopy output of data files and graphic displays through the use of a high speed DIABLO printer.
Freeze-fracture autoradiography. Progress towards a routine technique.
Authors: Schiller A , Taugner R , Rix E
Published: April 2011

Freeze-fracture autoradiography was introduced in 1976 as a new technique for the autoradiography of diffusible compounds at the electron microscope level. With the original approach coating of the frozen replicated specimens was performed in a cryostat at atmospheric pressure. Ice contamination of the specimen surface acting as an outstanding source of artifacts was thereby not excluded. With the use of a specially designed coating device and volatile spreading substances it was made possible to coat the frozen replicated specimens in the maintained vacuum of the freeze-fracture plant. In this complicated technique we have recently extended the freeze-fracture autoradiography to labeled frozen-dried "half" membranes of red blood cells.
Photometric methods in quantitation of light microscope radioautography.
Authors: Dörmer P
Published: April 2011

Photometric evaluation of autoradiographic grain densities can be performed according to various optical principles. In all instances the amount of light recorded should be a measure of the radioactive substance amount in the specimens. It is shown that grain densities suitable for light microscopic autoradiography are indeed linearly related to radiation exposure. Dependent on the photometric system used, there is a larger or smaller section of linear correlation of the photometric response with grain density. The advantages of incident light bright-field illumination for silver grain counting are discussed. Quantitation of substance amounts in autoradiographs depends on the use of radioactive standard sources. Two different approaches of quantitation are discussed. In 14C-autoradiography which can be applied for cell-kinetic studies, standard plates are used consisting of 14C-polymethylmethacrylate. Allowance has to be made for the different condition of radiation geometry of the cells and the standards. In 125I-autoradiography, 125I-labeled red cells are used as standards. This technique allows for quantitating the number of antibodies bound to individual cell surfaces.
Applications of stereology to the mechanics of soft tissues.
Authors: Vito R P
Published: April 2011

Quantification of microstructure in conjunction with mechanical testing and modeling may provide new insight into problems of soft tissue mechanics with potential clinical significance. Some general observations concerning this as well as brief discussions of specif examples are presented.
Morphometry of the effect of increased experience and training on synaptic density in area CA3 of the rat hippocampus.
Authors: Altschuler R A
Published: April 2011

A morphometric analysis of the effect of differential experiences on synapses was performed in area CA3 of the rat hippocampus. Two methods of quantitation that used a superimposed lattice grid were employed. In the first method squares overlying synapses were measured as a proportion of the total number of squares scanned; in the second method intersections of lattice lines with synapses were counted as a proportion of the total length of lines. Both measures showed that rats which were housed in an enriched environment and received 10 weeks of training had a significant increase in total area of synaptic contacts compared to both nonenriched controls and a croup with increased motor activity.
Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure.
Authors: Roels F , Goldfischer S
Published: April 2011

The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.
Double labeling immunohistochemical technique provides evidence of the specificity of glial cell markers.
Authors: Ghandour M S , Langley O K , Vincendon G , Gombos G
Published: April 2011

Sequential treatment of rat cerebellar slices with two antisera, each specific for a different glial cell marker, revealed, by indirect immunofluroescence combined with the immunoperoxidase method on the same tissue section, that carbonic anhydrase isoenzyme II (CA II) is exclusively localized in oligodendrocytes while the glial fibrillary acidic protein (GFA) is, as well established, a specific marker for astrocytes.
Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.
Authors: Simson J A , Spicer S S , Chao J , Grimm L , Margolius H S
Published: April 2011

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.
Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry.
Authors: Krishan A , Ganapathi R
Published: April 2011

The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracycline group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference of these compounds with the binding of propidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence of these antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use of these two methods for intracellular detection and quantitation of a number of important anthracyclines.
Separation of resting and proliferating granulocytic precursors.
Authors: Grogan W M , Scott R B , Collins J M
Published: April 2011

We have obtained cells in various stages of granulocytic development by a combination of isopycnic separation and electronic cell sorting. Not only were immature cells (blast cells, promyelocytes and myelocytes) separated from mature cells (bands and polys), but the immature cells were separated into proliferating (S + G2 + M) and resting (Go/G1) compartments of the cell cycle. This permits the study of the morphological and biochemical changes associated with development apart from those changes associated with proliferation.
NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney.
Authors: Wright J T , Corder C N
Published: April 2011

Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.
Localization of rhodopsin in isolated, osmotically intact rod outer segment discs.
Authors: Blaustein D I , Dewey M M
Published: April 2011

Localization of rhodopsin and its position in the membrane has been the subject of numerous studies. Most recently, immunocytochemical techniques have been employed to localize the opsin component of the molecule in in situ rod outer segments. Due to the problems inherent in localization procedures (penetration and mechanical interference) we have utilized isolated, osmotically intact rod outer segment discs in this study. Specific antibodies to chromatographically pure rhodopsin were prepared and enzymatically digested to their Fab components. The univalent Fab antibodies were conjugated to horseradish peroxidase and used to label the isolated rod outer segment discs. Discs treated with anti-opsin conjugate stained uniformly and heavily on their interdisc surfaces. Reaction product was also present on the intradisc surface in a thinner but still uniformly distributed layer. Controls treated with preimmune Fab - horseradish peroxidase conjugate showed no deposition of reaction product.
Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.
Authors: McElrath M J , Galbraith R M , Allen R C
Published: April 2011

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.
Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.
Authors: Arnold G , Liscum L , Holtzman E
Published: April 2011

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.
Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.
Authors: Chan A W , Perry S G , Burch H B , Fagioli S , Alvey T R , Lowry O H
Published: April 2011

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.
Distribution of glutamine synthetase in the rat central nervous system.
Authors: Norenberg M D
Published: April 2011

The results of a light microscopic immunohistochemical study of glutamine synthetase in rat nervous system are presented. In all sites studied the enzyme was confined to astrocytes. Except for trace amounts in ependymal cells, the enzyme was not observed in other cells of the nervous system including neurons, choroid plexus, third ventricular tanycytes, subependymal cells and mesodermally-derived elements. The intensity of astrocyte staining varied in different regions with the greatest degree noted in the hippocampus and cerebellar cortex while the least was noted in brain stem, deep cerebellar nuclei and spinal cord. The glutamine synthetase content correlated well with sites of suspected glutamergic activity in keeping with the view of a critical role of astrocytes in the regulation of the putative neurotransmitter glutamic acid.
The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.
Authors: Kirby W N , Parr E L
Published: April 2011

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.
Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.
Authors: Gonatas N K , Harper C , Mizutani T , Gonatas J O
Published: April 2011

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).
The cationic carbocyanine dyes Stains-all DBTC, and Ethyl-Stains-all, DBTC-3,3',9 triethyl.
Authors: Green M R , Pastewka J V
Published: April 2011

A simple method is presented for distinguishing two closely related metachromatic carbocyanine dyes: Ethyl-Stains-all, a triethyl dye, and Stains-all, a diethyl methyl dye. This has become important since one lot of the triethyl dye was distributed erroneously under the diethyl methyl label. The dyes differ in solubility and in differential staining of macromolecules. Studies performed with both dyes are summarized.
Nuclear labeling in immunoperoxidase studies of mouse tissue as detected by staining at acid pH.
Authors: Ripoche M A , Bernard O J , Bennett D
Published: April 2011

Several peroxidase-Ig conjugates were applied to sections of fixed mouse tissue. When the peroxidase staining reaction was done at pH 4.5 instead of pH 7.4, a striking reaction on nuclear membrane, chromatin, or chromosomes was observed. This staining was prevented by pretreatment of sections with DNase but not with RNase or after acid elution of histones. It is suggested that at acid pH a redistribution and binding DNA of oxidized chromogen or of a chromogen-conjugate complex to DNA may account for the results observed.
Alpha-bungarotoxin-horseradish peroxidase conjugate: preparation, properties and utilization for the histochemical detection of acetylcholine receptors.
Authors: Vogel Z , Towbin M , Daniels M P
Published: April 2011

A method is presented for the efficient conjugation of horseradish peroxidase to alpha-bungarotoxin. The 1:1 molar conjugate obtained is purified to completion by gel filtration on Sephadex G-100, followed by ion exchange chromatography on CM-Sephadex. The conjugate retains half of the activity of unmodified horseradish peroxidase and binds effectively to the nicotinic acetylcholine receptor of muscle. The conjugate is proven to be useful reagent for the histochemical staining of the receptor on muscle fibers for light and electron microscopy.
Cytochemical properties of mitochondria in the gastric parietal cell.
Authors: Sannes P L , Katsuyama T , Spicer S S
Published: April 2011

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.
Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst.
Authors: Howard R J , Battye F L , Mitchell G F
Published: April 2011

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.
Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP.
Authors: Rosenberg E M , LaVallee G , Weber P , Tucci S M
Published: April 2011

Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Conclusion: Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.
Potential inhibitors of rat tooth alkaline phosphatase studied by means of different histochemical techniques.
Authors: Larsson A , Hasselgren G
Published: April 2011

Two histochemical methods for demonstration of alkaline phosphatase activity, a lead pyrophosphate- anda naphtholphosphate technique, were compared. Since different results may be due to methodological differences as well as different enzyme activities, the enzymatic hydrolysis of the naphtholphosphate was visualized both by means of an azo-dye coupler and by lead-capturing of the liberated phosphate ion. Various potential inhibitors of alkaline phosphatase activity (diphosphonate, D-penicillamine, and sodium fluoride) were also tested. The use of diphosphonate and D-penicillamine resulted in inhibited or reduced staining, which could mainly be explained by an interference by these compounds with components in the incubation media rather than with the enzyme itself. The addition of sodium fluoride had no effect on the naphtholphosphate staining pattern irrespective of capturing method, whereas the odontoblastic pyrophosphate splitting alkaline phosphatase appeared to be sensitive to sodium fluoride, suggesting the presence of two alkaline phosphatases in odontoblasts.
On the immunocytochemical localization of the vasoactive intestinal polypeptide.
Authors: Larsson L I , Polak J M , Buffa R , Sundler F , Solcia E
Published: April 2011

The distribution of vasoactive intestinal polypeptide (VIP) immunoreactive nerves and endocrine cells in the gastrointestinal tract and pancreas of a number of mammalian and submammalian species has been examined in order to throw light on the exact localization of this peptide. Seven out of 8 VIP antisera demonstrated numerous nerve fibers in the gut, whereas one antiserum (TR2) revealed only scattered, few nerve fibers. The distribution of endocrine cells demonstrated by the different VIP antisera varied considerably. Thus, some antisera demonstrated only endocrine cells in the feline antrum, others only colonic endocrine cells and still others only endocrine cells of the upper gut and pancreas. The variability in staining pattern of endocrine cells as well as recent radioimmunological data makes it opportune to suggest that true VIP is a neuronal peptide and that endocrine cells store peptides resembling, but not being identical with, VIP (VIPoids).
Barrier filter for fluorescence microscopy of strongly autofluorescent plant tissues. Application to actin cables in Chara.
Authors: Nothnagle E A , Webb W W
Published: April 2011

A liquid barrier filter for use in fluorescence microscopy of strongly autofluorescent plant tissues is described. The filter consists of a methanol solution of cupric chloride and ferric chloride and isolates fluorescein fluorescence from the strong red autofluorescence of photosynthetic plant tissues. Subcortical actin cables in the giant alga Chara are being visualized through use of this filter together with heavy meromyosin labeling.
Cellular origins of different forms of gastrin. The specific immunocytochemical localization of related peptides.
Authors: Vaillant C , Dockray G , Hopkins C R
Published: April 2011

We have localized the antigenic determinants for the main forms of gastrin (big gastrin, G34, and little gastrin, G17) in hog antral mucosa using sequence specific antibodies and an indirect immunofluorescence technique. Populations of monospecific antibodies were obtained after affinity immunoadsorption to remove populations of unwanted specificity. The specificity of the purified antisera was established by direct binding of 125I labeled peptides to antisera at the same dilutions as those used in immunocytochemistry. The results indicate that in hog antral mucosa there is a single population of cells with the antigenic determinants of the C-terminal region of G17 and G34, the N-terminal region of G17, the N-terminal region of G34, and the intact G17 molecule. In duodenum there are cells with only C-terminal reactivity; since gastrin and CCK share a common C-terminal sequence it is concluded that this cell type contains CCK-like peptides rather than gastrin.
The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.
Authors: Ogawa H , Taneda A , Kanaoka Y , Sekine T
Published: April 2011

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.
Ultrastructural immunohistochemical localization of adrenocorticotropin and beta-lipotropin in the rat brain.
Authors: Pelletier G
Published: April 2011

In an attempt to identify the cells and organellel containing ACTH and beta-lipotropin in the rat brain, an immunocytochemical localization of these two peptides was performed at the electron microscopic level. Both ACTH and beta-lipotropin were localized in dense core vesicles of about 60-80 nm in diameter. Using serial sections, it has been possible to demonstrate that these peptides are contained not only in the same neuronal cell bodies, but also in the same dense core vesicles.
Accumulation of peroxidase in the cap rays of Acetabularia during the development of gametangia.
Authors: Menzel D
Published: April 2011

Accumulation of peroxidase was demonstrated by light and electron microscopy to occur in Acetabularia in certain regions of the cap rays in relation to the development of the gametangia (cysts). Peroxidase was found to be incorporated into special, cell wall-like obstructions that separate the cap rays from the stalk when the secondary nuclei have settled in the cap rays. It is assumed that peroxidase acts as an anti-microbial protectant of the gametangia.
A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue.
Authors: Weakley B S
Published: April 2011

Osmium-pyroantimonate solutions for the precipitation of cations are unsuitable for use with delicate mammalian oocytes. A variant of the pyroantimonate technique employing a mixture of pyroantimonate and glutaraldehyde has been found to give successful and repeatable results if a fixation time of 4 hr is used. Calcium-containing antimonate precipitates were localized principally in nuclei, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and cytoplasmic processes of both oocytes and follicle cells, and along the plasma membrane in small oocytes. Deposits were also concentrated around the periphery of lipid droplets in the follicle cells. The presence of calcium in the precipitates was confirmed by x-ray microprobe analysis.
Immunocytochemical localization of plasminogen activator on porcine kidney cell strain: LLC-PK1 (LP100).
Authors: Paul D C , Bobbitt J L , Williams D C , Hull R N
Published: April 2011

An antibody to plasminogen activator (PA) produced by the cultured cells of the pig kidney cell strain LLC-PK1 (LP100) was used to localize PA on the cell's free (unattached) surface. Localization was accomplished by the unlabeled antibody enzyme method (PAP) at the light microscopic level and at the electron microsopic level. Localization was commonly more intense at cell to cell junctions and was associated with blebs and vesiculation in this area. We are proposing that membrane shedding by blebs and vesiculation may be the mechanism of PA release in the LLC-PK1 (LP100) cell strain.
Histochemical detection of carbonic anhydrase with diemthylaminonaphthalene-5-sulfonamide.
Authors: Pochhammer C , Dietsch P , Siegmund P R
Published: April 2011

A new specific method for the detection of carbonic anhydrase, EC 4.2.1.1, in tissues is described. The reaction of carbonic anhydrase with dimethylaminonaphthalene-5-sulfonamide (DNSA) forms a highly fluorescent complex. The specificity of the method is proved by the quenching of this fluorescence with ethoxzolamide (6-ethoxybenzothiazole-5-sulfonamide). The difference in the wavelength makes it possible to absorb the fluorescence of the unbound dimethylaminonaphthalene-5-sulfonamide by filters. Kidney, proventriculus, and bone from chicken have been examined. Carbonic anhydrase has been detected in the cytoplasm of the columnar lining cells, proximal tubule cells, and osteoclasts.
A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.
Authors: White D L , Mazurkiewicz J E , Barrnett R J
Published: April 2011

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.
Ultrastructural localization of sulfated complex carbohydrates with a modified iron diamine procedure.
Authors: Sannes P L , Spicer S S , Katsuyama T
Published: April 2011

A thiocarbohydrazide-silver proteinate (TCH-SP) sequence was applied to thin sections of specimens that had been reacted with the high iron diamine (HID) method for ultrastructural localization of sulfated complex carbohydrates. The exposure to TCH-SP enhanced the electron opacity of HID-reactive sites and increased the sensitivity of the procedure. This held true for HID-reacted specimens whether or not they had been post-treated with osmium tetroxide. However in those not postosmicated after HID, the contrast and specificity appeared superior, as sites of osmiophilia were densified equally in specimens exposed to HID, and unexposed controls, by the final osmium tetroxide-TCH-SP sequence. Staining of immature granules of developing polymorphonuclear neutrophils by HID was intensified by the post-treatment with TCH-SP. In addition, granules of blood mononuclear leukocytes and heterophagosomes of peritoneal macrophages revealed HID affinity and hence content of sulfated mucosubstance that was not evident without the TCH-SP steps. Control procedures which entailed initial exposure of the specimen to FeCl3 or MgCl2 solutions and treatment of thin sections with TCH-SP failed to impart density to these sites.
Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.
Authors: Limacher W , Wild P , Manser E , Lutz H
Published: April 2011

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.
The use of lectins and cholera toxin for the detection of surface carbohydrates of cultured neurons and neuroblastoma.
Authors: Gonatas N K
Published: April 2011

Conjugates of horseradish peroxidase with the lectins ricin (d-galactose), wheat germ agglutinin (N-acetylglucosamine), phytohemagglutinin (N-acetylgalactosamine), and with cholera toxin (GM1 ganglioside) were used for a cytochemical detection of corresponding termin al carbohydrates, or glycolipids on cell surfaces of cultured neurons and neuroblastoma cells. Cells were labeled at 4 degrees C with the above ligands and their adsorptive endocytosis was studied after incubations at 37 degrees C in a medium free of ligand. Peroxidase was detected by the method of Graham and Karnovsky (J. Histochem. Cytochem. 14:291, 1966). Lectins and cholera toxin underwent endocytosis in cisternae and vesicles of GERL (Golgi-Endoplasmic Reticulum-Lysosome). We suggest that GERL is the primary ercipieint of adsorptively endocytosed plasma membrane "receptor"-ligand complexes which are thus degraded or possibly reutilized (recycling). Wheat germ agglutinin-horseradish peroxidase conjugates used in vivo for studies of retrograde axonal transport were significantly more sensitive than free horseradish peroxidase.
Subcellular localization of complex carbohydrates in rat macrophages and monocytes.
Authors: Sannes P L , Eguchi M , Spicer S S
Published: April 2011

Methods for visualization of complex carbohydrates ultrastructurally were employed to study specific organelles of the rat monocyte and macrophage. Vicinal glycols of glycoconjugates were demonstrated with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) postembedding sequence and acid groups were delineated by the dialyzed iron (DI) and high iron diamine (HID) preembedding techniques. Lysosomal bodies were generally found reactive with all three methods, although those of monocytes from the bone marrow and peripheral blood were notably lacking in acidic groups. The Golgi complex was consistently PA-TCH-SP-reactive, as were associated vesicles and occasional cisternal expansions, possibly related to GERL. Numerous cytoplasmic vesicles and small granulated structures and cisternae of the rough endoplasmic reticulum were also PA-TCH-SP-reactive.
Cytochemical characterization of secretory and cell surface glycoconjugates by light and electron microscopy.
Authors: Spicer S S , Sannes P L , Katsuyama T
Published: April 2011

Lectin methods have increased the capacity for histochemical characterization and differentiation of glycoproteins and have demonstrated, for example, greater reactivity of gastrointestinal than of respiratory tract secretions with the periodate-concanavalin A-horseradish peroxidase method for localizing mannose-rich glycoprotein. Application of a battery of ultrastructural cytochemical methods with specificity for the constituents characteristically present in the complex carbohydrates provides knowledge of the distribution of the various recognizable types of glycoconjugates in tissues and cells showing, for example, marked differences in glycoconjugates of the apical compared with the basolateral plasmalemma in a given cell type and differences between apical plasmalemmas or basement membranes of different cell types. Such information raises questions as to the biologic significance of the different complex carbohydrates in various sites and, hopefully, will lead to a clearer understanding of their physiologic roles.
Clinical applications of the enzyme labeled antibody method. Immunoperoxidase methods in diagnostic histopathology.
Authors: Bosman F T , Nieuwenhuijzen Kruseman A C
Published: April 2011

Histological criteria for the definition of disease entities have largely been established with light microscopy of conventionally stained and routinely processed tissue sections. More or less specific histochemical staining procedures and more recently enzyme-histochemical and quantitative histo- and cytochemical techniques in some cases provided additional criteria. In the last decade, however, the introduction of immunofluorescence and more recently the different immunoperoxidase methods have significantly influenced the scope of contemporary histopathology. Especially, the possibility to use immunoperoxidase methods on routinely processed tissue specimens has offered new dimensions in diagnostic pathology. These methods proved of particular importance for: 1) The development of new criteria for diagnosis, prognosis and treatment (e.g. immunological classification of lymphoma; plasmacell typing in intestinal inflammatory conditions; human chorionic gonadotropin and alpha-fetoprotein in germ cell tumors of the testis). 2) The possibility of etiological diagnosis (e.g. the recognition of hepatitis B viral antigens in liver biopsy specimens; histological typing of causative micro-organisms in inflammatory conditions). 3) The recognition of disease entities that were hitherto unrecognized (e.g. hyperplasia of parafolicular C-cells in the thyroid in multiple endocrine neoplasia syndromes; gastric G-cell hyperplasia as a variant of Zollinger-Ellisons syndrome). 4) Functional analysis of tissue components (e.g. hormone content of pituitary and pancreatic adenomas; cytoplasmic differentiation produces in "undifferentiated" tumors). It can be expected that immunoenzymehistochemistry will soon play a major role in routine diagnostic histopathology.