bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations.
Authors: Hickey W F , Lee V , Trojanowski J Q , McMillan L J , McKearn T J , Gonatas J , Gonatas N K
Published: April 2011

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.
An avidin-biotin-peroxidase method for Fc receptors on macrophages isolated from and in sections of rat lung.
Authors: Elner V M , Hass A J , Davis H R , Glagov S
Published: April 2011

Receptors for the Fc region of immunoglobulin G (Fc receptors) were detected on pulmonary macrophages by adapting an avidin-biotin-peroxidase technique to isolated cells and sections of rat lung. After incubation with soluble rabbit immunoglobulin G (IgG), surface bound IgG was identified consistently and reproducibly on glass-adherent pulmonary macrophages and on macrophages in tissue sections made from incubated lung slices. Control experiments indicated that binding was specifically mediated by surface Fc receptors. This method may be useful for identifying macrophages in intact tissues.
Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunocytochemically identified neuropeptide.
Authors: Oldfield B J , Hou-Yu A , Silverman A J
Published: April 2011

In order to extend the information obtainable from ultrastructural studies of synaptic connectivity using either horseradish peroxidase tracing or immunocytochemistry alone, we have developed a method of combining these two procedures. Thus it has been possible to examine the characteristics of axon terminals of known origin forming synaptic contacts with cells of identified neuropeptide content.
Nuclear localization of lactoferrin in the human granulocyte: Artifact incurred during slide preparation.
Authors: Briggs R C , Montiel M M , Wojtkowiak Z
Published: April 2011

Within the blood cells, lactoferrin is found only in the late stage neutrophilic granulocytes. Lactoferrin first appears in these cells during the myelocyte stage of development coincidentally with the specific or secondary granules. Most investigators report a cytoplasmic immunocytochemical localization reaction within the granulocyte. However, others have observed a prominent nuclear localization reaction. Treating the cells with certain fixatives was shown to prevent the relocation of lactoferrin from the cytoplasm to the nucleus when the localization was done on granulocytes prepared by smearing. The present study demonstrated that the relocation of lactoferrin is only a problem when cells were smeared or cytocentrifuged onto slides or fractionated for the purpose of isolating cellular organelles. Under these conditions the selection of fixative is an important consideration. Exposing isolated lactoferrin to a fixative effective in retaining lactoferrin in the cytoplasm of granulocytes smeared on slides did not alter a number of its physical properties. The results suggest that maintenance of the normal cytoarchitecture or effect of fixative on other cellular components prevents the relocation of lactoferrin within the cell during tissue processing and the direct action of fixation on lactoferrin is probably not responsible for this effect.
Identification of the APUD endocrine cells of rat fundic mucosa by means of combined amine fluorescence and electron microscopy.
Authors: Rubin W , Schwartz B
Published: April 2011

Rat fundic mucosa contains numerous APUD endocrine cells that can take up 5-hydroxytryptophan (5-HTP) and decarboxylate it to 5-hydroxytryptamine (serotonin), detectable by its formaldehyde-induced yellow fluorescence. To identify these cells by electron microscopy, pieces of rat gastric mucosa were incubated with DL-5-HTP. Some specimens were fixed in 4% formaldehyde-0.5% glutaraldehyde, while others were frozen, freeze-dried, and exposed to paraformaldehyde vapors. Thick sections of the Epon-embedded specimens were examined and photographed by fluorescence microscopy, and fluorescing and nonfluorescing cells were identified by electron microscopy in serial thin sections. Control specimens, not incubated with 5-HTP, revealed fluorescence of only the mast cells and EC cells, which were abundant in the pylorus, but rare in fundic mucosa. Specimens incubated with 5-HTP also exhibited numerous yellow fluorescent endocrine cells in fundic mucosa, which cells were found to be the ECL cells; the A-like cells did not fluoresce. In pyloric mucosa, many G and D (D1) cells also exhibited weak or moderate fluorescence after 5-HTP incubation. Thus, this study supports the contention of previous radioautographic studies that the ECL, but not the A-like, cells are the APUD endocrine cells of rat fundic mucosa, that G and D cells also possess some APUD activity, and that EC cells represent the enterochromaffin cells, which normally synthesize and store demonstrable quantities of serotonin.
Use of acridine orange for histologic analysis of the central nervous system.
Authors: Mai J K , Schmidt-Kastner R , Tefett H B
Published: April 2011

The application of the fluorescent dye acridine orange (AO) to the staining of histologic sections of the brain, and its use for automatic cyto- and histophotometric evaluation are described and compared with the results obtained using cresyl violet. The most suitable procedure for aldehyde-fixed brain tissue, embedded in paraffin and sectioned at 5 micron, proved to be treatment of the sections with an aqueous solution of AO (1:50,000) at pH 1.2 for 30 min, followed by rinsing in distilled water for 10 min. This procedure revealed the morphology in a highly acceptable manner, clearly differentiating various cell components; its characteristics included exact reproducibility and high contrast. The degree of fading was calculable, with a very gradual decrease in fluorescent intensity. The AO procedure appears to be compatible with most other staining procedures that do not rely on the same binding mechanisms. Thus, AO staining has two advantages over the classical cytoarchitectural staining methods: first, it is more suitable for automated morphometric analysis, and second, it can be used in conjunction with immunologic and other techniques.
An immunologic study of the secretory products of rat Clara cells.
Authors: Singh G , Katyal S L
Published: April 2011

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.
Immunohistochemical localization of galactosyltransferase in the normal human ovary and fallopian tube.
Authors: Davis B W , Berger E G , Locher G W , Zeller M , Goldhirsch A
Published: April 2011

Galactosyltransferase (UDP-galactose: 2-acetamido-2-deoxy beta-D-glucopyranose beta-(1-4) transferase) in human tissue specimens from ovaries and the corresponding fallopian tubes was localized immunohistochemically for light microscopy. An affinity-purified rabbit anti-human milk galactosyltransferase antibody was used. Intracellular galactosyltransferase was found to be localized to the juxtanuclear (Golgi) region of the secretory cells of the fallopian-tube epithelium and to the ovarian stromal cells involved in steroid-hormone production. Cell-surface galactosyltransferase was localized to ciliated cells of the fallopian-tube epithelium. During the follicular phase of the menstrual cycle, galactosyltransferase was found only in the Golgi regions of theca interna cells of the ovarian graafian follicle, and in the fallopian tube was found predominantly on the cilia of epithelial cells. During the luteal phase, galactosyltransferase was abundant in the Golgi regions of granulosa lutein cells of the corpus luteum, and was predominant in the secretory cells of the tubal epithelium. Galactosyltransferase was not detected on the mesothelial ovarian surface. The results demonstrate that the cellular distribution and location of galactosyltransferase correlates with phenotypic differentiation and varies during the human female hormonal cycle.
Immunoreactive kallikrein localization in the rat kidney: an immuno-electron-microscopic study.
Authors: Figueroa C D , Caorsi I , Subiabre J , Vío C P
Published: April 2011

The cellular and subcellular localization of immunoreactive kallikrein was studied in the rat kidney using the peroxidase-antiperoxidase (PAP) method for the electron microscope. The effect of various tissue-processing protocols on ultrastructural preservation and immunocytochemical staining was evaluated by fixing kidneys with four different mixtures. The tissues were immunostained and further stained with OsO4 or silver methenamine. The best ultrastructural and immunocytochemical staining was obtained with Zamboni's-glutaraldehyde fixative. The kallikrein-immunoreactive cell type was identified, according to its localization and ultrastructural features, as the connecting-tubule cell. Immunoreactive kallikrein was concentrated mainly in the upper one-third of the cell and at both sides of the nuclei, and to a less extent was associated with the plasma membranes and basolateral infoldings. The immunoreactivity was related to free polyribosomes, the rough endoplasmic reticulum (RER), and the Golgi complex, suggesting that kallikrein is actively synthesized in this particular type of cell.
Lactate dehydrogenase in developing rat oral epithelium.
Authors: Sjögren S
Published: April 2011

Freeze-dried sagittal, whole-body sections of 10-day-old rats were incubated for lactic dehydrogenase (LDH) using different media in the presence of the inhibitors urea and fluoropyruvate. Phenazine methosulfate (PMS) and menadione, which are regularly used in current histochemical media and are believed to promote the demonstration of LDH activity, were also added and shown to be insufficient for the demonstration of total LDH activity, and PMS even seemed to have an inhibitory effect on LDH activity in oral epithelium. However, cumulated data from the different incubations show that the oral epithelium of developing rats may contain two different types of LDH, one in the basal cells with possibly aerobic characteristics, and another in the spinosum/granulosum cells with anaerobic characteristics.
Contrast enhancement of the brownish horseradish peroxidase-activated 3,3'-diaminobenzidine tetrahydrochloride reaction product in black and white photomicrography by the use of interference filters.
Authors: Møller M , Glistrup O V , Olsen W
Published: April 2011

An interference filter combination for contrast enhancement of the brownish, peroxidase-activated, 3,3'-diaminobenzidine tetrahydrochloride reaction product has been developed. This filter combination allows transmission of light in two narrow bands, representing those of the primary colors green (480-500 nm) and red (575-585 nm) as well as a broader band between 385-430 nm. The visual contrast enhancement of horseradish peroxidase-containing neurons caused by the interference filters was excellent. In particular, intracellular details were prominent in the presence of the interference filters compared with different blue absorption filters. The contrast enhancement was evaluated by microdensitometry of an Agfapan 25 emulsion onto which peroxidase-containing neurons in noncounterstained sections were exposed. The microdensitometry showed a neuron to background contrast enhancement of 30% compared to neutral gray filters and of 11% compared to a Kodak Wratten 80A blue absorption filter. The interference filters are available from the Laboratory of Technical Optics, the Technical University of Copenhagen, Building 307, Lundtoftevej 100, DK-2800 Lyngby, Denmark (order: DAB-interference filters, 30 mm). The interference filter principle can be used for contrast enhancement of other compound colors.
Cytochemical detection of hyaluronidase activity in single human and mouse sperm by an improved substrate-film technique.
Authors: Waibel R , Ginsberg L C , Ficsor G
Published: April 2011

A substrate-film method is described that allows the detection of hyaluronidase activity in nearly 100% of single human and mouse sperm. The level of hyaluronidase activity as determined by halo diameters was greater in mouse than in human sperm. This simple method may have use as a screening method for identifying compounds that cause developmental or genetic defects in male germ cells, or for the diagnosis of infertility due to decreased hyaluronidase activity.
Monoclonal antibody LICR-LON-E36 recognizes gonadotrophs in female rat pituitaries.
Authors: Monaghan P , Sappino A P , Roberts J D , Knight M A , Ellis J , Nakajima T
Published: April 2011

The distribution of the antigen localized by monoclonal antibody LICR-LON-E36 has been studied by means of light and electron microscopical immunocytochemical procedures on resin-embedded pituitaries from male and female rats. Using both immunoperoxidase and immunogold labeling techniques, the localization of LICR-LON-E36 has been compared with that obtained with antibodies against the beta subunit of rat luteinizing hormone (beta LH) and human follicle stimulating hormone (beta FSH), porcine adrenocroticotropic hormone (ACTH) and rat S-100. LICR-LON-E36 is localized in a portion of beta LH-containing cells in female rat pituitaries and where present both antigens are localized within the same storage granules. LICR-LON-E36 is rarely detectable within beta LH-containing cells of male rat pituitaries that also stain positively with anti-beta FSH antibodies. ACTH and S-100 were localized within different cell populations.
A monoclonal antibody detecting neural and neuroendocrine differentiation.
Authors: Sappino A P , McIlhinney R A , Ellison M , Monaghan P , Neville A M
Published: April 2011

A monoclonal antibody, named LICR-LON-E36, has been produced to the acidic fraction of soluble protein extracts of human brains. Using immunocytochemistry, it stained normal peripheral nerves, adrenal medulla, pancreatic islets, and enterochromaffin cells, in addition to recognizing an intracytoplasmic epitope in phaeochromocytomas, carcinoid tumors, some small-cell anaplastic lung, and a few breast carcinomas. Microenzyme-linked immunosorbent assay and electrophoretic nitrocellulose blotting experiments confirmed that the antibody is not directed at neuron-specific enolase. Its potential use as an immunological probe for neural and neuroendocrine differentiation is discussed.
Evaluation of potassium pyroantimonate/sucrose/glutaraldehyde concentration and incubation time as essential variables for localizing calcium bound to organic compounds in epiphyseal chondrocytes.
Authors: Kashiwa H K , Thiersch N J
Published: April 2011

The solution chemistry of potassium pyroantimonate (PA) was clarified, enabling preparation of stable concentrated PA solutions. The soluble conjugate base of PA determines the pH of the solution; therefore, the pH reflects the maximum amount of conjugate base dissolved ("saturated") in solution. Spontaneous precipitation occurs just below the "saturation" pH. Sucrose (S) stabilizes the conjugate base exponentially, with precipitation of the sucrose-stabilized PA occurring far below the saturation pH in alkaline solution and at the saturation pH in neutral solution. Using glutaraldehyde (G) at 4% concentration to preserve the cells and using 2-7% PASG solutions at pH 8.5, calcium bound to organic compounds was localized in the hypertrophied chondrocytes at the zone of calcification with optimal reaction. The minimal incubation time for optimal reaction increased with decreasing PA concentration; from 2 hr for 7% PA to 36 hr for 2% PA. With optimal reaction, electron-dense granules were localized in the nucleus, nuclear membrane, endoplasmic reticulum cisterns, mitochondria, Golgi apparatus, intercisternal matrix, plasma membrane, intercellular matrix, and calcospherules. Ethyleneglycoltetraacetic acid immersion and X-ray microprobe studies of the electron-dense granules indicated calcium as the predominant cation. Control tissues with implanted tricalcium phosphate crystals, and reaction kinetic between PA and cellular calcium, indicated that the calcium localized was not due to translocation but to calcium bound to organic compound that is synthesized by the hypertrophied cells in the zone of calcification.
A double-immunostaining procedure using colloidal gold, ferritin, and peroxidase as markers for simultaneous detection of two hypophysial hormones with the electron microscope.
Authors: Doerr-Schott J , Lichte C M
Published: April 2011

For simultaneous ultrastructural localization of intracellular peptides, protein A-gold techniques or immuno-gold techniques have generally been applied. The present study reports a double immunostaining procedure for simultaneous visualization of two hypophysial hormones (prolactin and corticotropin) on a single ultrathin section of the pars distalis of an amphibian. Prolactin and corticotropin antisera were respectively raised in guinea pigs and rabbits and were applied simultaneously to ultrathin sections. Antigenic binding sites were detected under the electron microscope using differently labeled species-specific secondary antisera raised in goats or sheep. Three labels (gold particles, ferritin, peroxidase) were checked for double labeling. The combinations investigated were: 1) two gold preparations or IgG-gold labeled with different-sized gold particles; 2)IgG-gold and IgG-ferritin; 3) IgG-gold and IgG-PAP (peroxidase-antiperoxidase). The double-immunostaining procedures described here have proved useful in the simultaneous ultrastructural localizations of two intracellular antigens on a single tissue section. These procedures constitute a basis for the development of triple immunostaining methods.
Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes.
Authors: Roth J , Lucocq J M , Charest P M
Published: April 2011

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.
Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification.
Authors: Panayotacopoulou M T , Issidorides M R
Published: April 2011

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.
Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2.
Authors: Allison D C , Meyne J , Ridolpho P F , Bose K , Chakerian M , Robertson J
Published: April 2011

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.
Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy.
Authors: Altman L G , Schneider B G , Papermaster D S
Published: April 2011

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.
Myoglobin levels in individual human skeletal muscle fibers of different types.
Authors: Nemeth P M , Lowry O H
Published: April 2011

An attempt was made to determine the relationship of myoglobin content to specific fiber types in human muscle. Biopsies were obtained from biceps brachii, vastus lateralis, and gastrocnemius muscles of untrained subjects and from the vastus lateralis muscle of a highly trained athlete at peak training and at intervals of no training (detraining). Individual muscle fibers were assayed, by quantitative microanalytical methods, for myoglobin, lactate dehydrogenase, malate dehydrogenase, citrate synthase, beta-hydroxyacyl-coenzyme A dehydrogenase, and adenylokinase activities all on the same fiber. The enzyme levels were used to classify the fibers into type I or II. The results show that the content of myoglobin in human muscle does not differ greatly between fiber types in contrast to other species. The type II fibers contained, on the average, at least two-thirds as much myoglobin as type I fibers. The concentration of myoglobin did not change in either fiber type during detraining (84 days), despite marked changes in lactate dehydrogenase, adenylokinase and the three oxidative enzymes.
Distribution of calcium during interphase and mitosis as observed by ion microscopy.
Authors: Chandra S , Harris W C , Morrison G H
Published: April 2011

The ion microscope, based on secondary ion mass spectrometry, has been used to demonstrate the distribution of calcium in the root tip cells of two plant species, Allium cepa and Vicia faba. Interphase nuclei showed higher intensities of calcium than cytoplasm, while nucleoli exhibited higher calcium intensities than the rest of the nucleoplasm. The chromosomes showed high intensities of calcium at all stages of mitosis. Calcium was also detected in the cell plate and phragmoplast region of dividing cells. It appears that during prophase calcium concentrates in the condensing chromosomes, and during telophase it is transferred to nucleoli. These observations suggest that chromosomes may serve as a reservoir of calcium during mitosis.
Immunocytochemical localization of glucocorticoid receptors in midgestation murine embryos and human embryonic cultured cells.
Authors: Kim C S , Lauder J M , Joh T H , Pratt R M
Published: April 2011

Glucocorticoid receptors have been localized immunocytochemically in the developing mouse secondary palatal shelves and in cultured human embryonic palatal mesenchyme cells. In the midgestation embryo, receptors are found in the highest concentration in the palatal mesenchymal cells, suggesting that they play a major role in normal development as well as in glucocorticoid-induced cleft palate. The presence of these receptors in cultured human embryonic palatal cells also suggests that development of the human secondary palate may be dependent on glucocorticoids.
Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry.
Authors: Rye D B , Saper C B , Wainer B H
Published: April 2011

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.
Identification of protease isozymes after analytical isoelectric focusing using fluorogenic substrates impregnated into cellulose membranes.
Authors: Smith R E
Published: April 2011

A technique to characterize active isozymes of proteases is described. After isoelectric focusing in an ultrathin gel, a cellulose diacetate or hydrate membrane previously impregnated with media containing a fluorogenic substrate is applied to the surface of the gel. Low-molecular-weight peptide inhibitors can also be added to the membrane to enhance specificity. The technique shows excellent reproducibility, specificity, and sensitivity, and has promise for the study of isozymes of proteases in tissues and body fluids in normal and pathological states.
Localization of human muscle carbonic anhydrase isozymes using immunofluorescence.
Authors: Moyle S , Jeffery S , Carter N D
Published: April 2011

Human muscle sections were examined for the presence of carbonic anhydrase using fluorescent second antibody and antisera specific for the three isozymes, carbonic anhydrase I (CAI), carbonic anhydrase II (CAII), and carbonic anhydrase (CAIII). CAIII was present in all fibers, CAII only in the connective tissue, and CAI showed a weak association with the sarcolemma.
Hypotensive effects in cats caused by horseradish peroxidase mediated by metabolites of arachidonic acid.
Authors: Sjaastad O V , Blom A K , Haye R
Published: April 2011

Horseradish peroxidase (HRP) given intravenously was shown to possess a marked hypotensive effect in cats. The effect was not noticeably influenced by antihistamines. Indomethacin and acetylsalicylic acid completely blocked the hypotensive effect of HRP. It is concluded that the effect of HRP on blood pressure is due to stimulation of the synthesis of metabolites of arachidonic acid.
Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).
Authors: Cordell J L , Falini B , Erber W N , Ghosh A K , Abdulaziz Z , MacDonald S , Pulford K A , Stein H , Mason D Y
Published: April 2011

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").
Glucose-6-phosphatase as a cytochemical marker of endoplasmic reticulum in human leukocytes and platelets.
Authors: Nichols B A , Setzer P Y , Bainton D F
Published: April 2011

Leukocytes and platelets, freshly isolated from normal human blood, were tested cytochemically for glucose-6-phosphatase (G-6-Pase) by a modified Wachstein-Meisel method. The enzyme was present in the endoplasmic reticulum (ER) and perinuclear cisternae of all five types of leukocytes and in the ER of platelets. The reaction product from the cytochemical test distinguished the ER from other intracellular membrane-limited cisternae (i.e., the smooth pinocytic tubules of monocytes and the surface-connected canalicular system of platelets) and thus is a valuable marker of the ER. The cytochemical test also showed that the ER of polymorphonuclear leukocytes (PMN), usually obscured by abundant granules in cells prepared for morphological examination, is more extensive than formerly appreciated. This is the first demonstration of G-6-Pase in human leukocytes. Its precise role in leukocyte metabolism can now be investigated.
The connective tissue of the rat lung: electron immunohistochemical studies.
Authors: Gil J , Martinez-Hernandez A
Published: April 2011

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.
Visualization of peptide-immunoreactive processes on serotonin-immunoreactive cells using two-color immunoperoxidase staining.
Authors: Hancock M B
Published: April 2011

The sequential application of the peroxidase-antiperoxidase (PAP) technique with nickel-intensified DAB and with DAB alone was used to visualize black peptide-immunoreactive endings on amber serotonin-immunoreactive cells in 1-2-micron paraffin sections of the hamster medulla. Met-enkephalin- and substance P-positive terminals were present on serotonin cells in the raphe nuclei, the ventral reticular formation, and in the nucleus interfascicularis hypoglossi. The presence of enkephalin-immunoreactive endings on medullary serotonin-immunoreactive cells correlates with the analgesia and autonomic changes that result from the application of morphine or met-enkephalin to the medulla.
Monoclonal antibodies against amyloid fibril protein AA. Production, specificity, and use for immunohistochemical localization and classification of AA-type amyloidosis.
Authors: Linke R P
Published: April 2011

Monoclonal antibodies against amyloid fibril protein AA were produced by cell fusion of murine P3 X 63-Ag8.653 myeloma cells with spleen cells of immunized Balb/c mice. To increase immunogenicity, protein AA was coupled to horseradish peroxidase (HRP) or human high molecular weight kininogen (HMWK). Using micro-ELISA (enzyme-linked immunosorbent essay) seven hybridoma cell lines secreting antibodies that specifically bind to protein AA have been selected and cloned. When applied to formalin-fixed paraffin sections of a variety of different amyloid types using immunoperoxidase methods, five monoclonal antibodies bound specifically and strongly to amyloid only of the AA type. Since a series of different AA-amyloids could be stained, these reagents may be used to routinely diagnose AA-amyloidosis in tissue sections. A monoclonal antibody against HRP has also been produced that has been utilized to develop a monoclonal peroxidase-antiperoxidases (PAP) complex. When three immunoperoxidase methods were compared, the sensitivity of a conventional rat PAP was comparable to the monoclonal PAP complex, but the latter was easier to handle. Both methods were more sensitive than the indirect immunoperoxidase technique.
Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells.
Authors: Berrih S , Savino W , Azoulay M , Dardenne M , Bach J F
Published: April 2011

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.
Lymphocyte enumeration: a comparison between a modified avidin-biotin-immunoperoxidase system and flow cytometry.
Authors: Paradis I L , Merrall E J , Krell J M , Dauber J H , Rogers R M , Rabin B S
Published: April 2011

The reliability and sensitivity of an indirect avidin-biotin-peroxidase (ABC) procedure for enumerating lymphocyte subpopulations was compared to flow cytometry (FC) employing direct immunofluorescence. Lymphocytes were enumerated by two different methods. For counting method I, which is the method of conventional FC, the number of immunostained lymphocytes was compared to the total number of lymphocytes present. The ABC procedure by method I detected a greater proportion of immunostained lymphocytes for all subsets tested than did FC. By counting method II, where the number of immunostained lymphocytes is compared to the total number of cells present, the ABC analysis still detected more total T cells than FC but the results for the two analyses were similar for T helper and T suppressor cells. Thus, the ABC technique appears to be a valid method for enumerating T lymphocyte subsets. Furthermore, as compared to FC, it offers the advantages of reduced cost, simplicity of understanding and performance, need for fewer cells, and a permanent record of lymphocyte staining. For these reasons, we feel that the ABC technique will enjoy widespread application for the identification of lymphocyte membrane antigens.
Immunocytochemical localization of mouse placental lactogen in the mouse placenta.
Authors: Hall J , Talamantes F
Published: April 2011

The localization of mouse placental lactogen (mPL) in the mouse placenta has been examined with the avidin-biotin-peroxidase complex (ABC) technique. Staining was observed to occur in the basal zone of placentae from days 10, 12, 15, and 18 of gestation. Within the basal zone, the most prominent staining was localized to the cytoplasm of the giant cells. Within the labyrinth, staining was observed in the giant cells in day-15 and day-18 placentae, with a greater number of stained cells being observed in the latter.
Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity.
Authors: Wainer B H , Rye D B
Published: April 2011

Localization of choline acetyltransferase (ChAT) immunoreactivity in rodent brain (AI Levey, DM Armstrong, SF Atweh, RD Terry, BH Wainer: J Neurosci 3 1, 1983) with a monoclonal antibody (Ab8) has been previously reported. Now a procedure for combining ChAT immunohistochemistry with retrograde tracing for the purpose of mapping cholinergic pathways is presented. Rats were injected with horseradish peroxidase-wheat germ agglutinin in the tongue and cerebral cortex. Sections from their perfusion-fixed brains were reacted with 3,3'-diaminobenzidine (DAB)/H2O2/cobalt acetate followed by ChAT localization with monoclonal antibody Ab8 using the peroxidase-antiperoxidase method and visualization using DAB/H2O2. Double-labeled cells were visualized with black punctate staining (retrograde tracer) on a diffuse brown cytoplasmic background (ChAT immunoreactivity) in the hypoglossal nucleus and ventral telencephalon (substantia innominata-nucleus basalis). Conditions of fixation, histochemistry, and immunohistochemistry that contribute to optimal resolution for this procedure are discussed.
Detection of herpes simplex virus infection using glucose oxidase-antiglucose oxidase immunoenzymatic stain.
Authors: Gay H , Clark W R , Docherty J J
Published: April 2011

Herpes simplex virus (HSV) infected cells have been detected in tissue culture and human cell specimens by an immunoenzymatic staining method using the fungal enzyme glucose oxidase. Infected cells from culture or human specimens appear as dark blue, brown, or red, depending on the tetrazolium salt used in the disclosing reaction, with virtually no staining of uninfected cells. The specificity and sensitivity of this method and of the more commonly used immunoperoxidase method are comparable, but the immunoglucose oxidase method avoids the problems of nonspecific staining by the endogenous peroxidase present in mucosecretions and inflammatory cells. Staining time can be reduced up to 40% of that necessary for the unlabeled immunoperoxidase procedure without compromising the quality of staining results.
Immunohistochemical demonstration of surface antigen of human lymphocytes with monoclonal antibody in acetone-fixed paraffin-embedded sections.
Authors: Tanaka M , Tanaka H , Ishikawa E
Published: April 2011

Although the majority of reported studies have used fresh-frozen sections in detecting surface antigen of lymphocytes in tissue via monoclonal antibody, detailed histological figures can not be obtained by this method. Nor can the antigenicity be preserved for any length of time. A new method for detecting the surface antigen of lymphocytes using fixed and embedded material is presented. Human spleens were fixed in cold acetone, embedded in low melting point paraffin wax, and the thin sections treated with hyaluronidase. Anti-T lymphocyte monoclonal antibody (anti-Leu-1, anti-Leu-2, anti-Leu-3) and anti-HLA-DR were applied on these sections, and the antigen was detected by the ABC (avidin-biotin-peroxidase complex) method. The results were then compared with those of fresh-frozen sections. There was no great difference in detecting T and B cells or their subsets, but the histological figures were substantially better preserved in sections prepared by the present method. Furthermore, the antigenicity was retained in the materials fixed and embedded for more than two years.
Cytokinetics of subpopulations in mixed polyploid tumors by television imaging. III. 3H-thymidine incorporation by ploidy subpopulations--control of 3H-absorption and emulsion efficiency in autoradiography.
Authors: Sklarew R J
Published: April 2011

An approach is presented for determining relative rates of 3H-thymidine incorporation in S-phase for discrete ploidy subpopulations that coexist in heteroploid tumors. The analysis is facilitated by an automated imaging methodology developed with a Quantimet 720 System for simultaneous determination of Feulgen-stained DNA content and grain counts of 3H-thymidine-labeled cells in Feulgen-stained autoradiographs (Sklarew RJ: J Histochem Cytochem 30:35, 30:49, 1982). The experimental design involves 3H- and 14C-thymidine double labeling in the presence of Colcemid. Data from heteroploid rat sarcoma cultures illustrate the profound influence of differential 3H absorption and relative emulsion efficiency. These factors are controlled, respectively, through measurements of mean nuclear optical density and grain density. This is essential for a valid comparison of grain counts between cells that differ in their autoradiographic geometry. Thus, relative DNA specific activity may be assessed in individual cells. The protocol and analysis supports a simultaneous evaluation of the S-phase ploidy composition and S-emptying profile, as presented in a companion article (Sklarew RJ: J Histochem Cytochem 32:413, 1984).
A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses.
Authors: Morrison-Graham K , O'Lague P H
Published: April 2011

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.