bluuebunny/crossref_metadata_2025 huggingface dataset:
An unlabeled antibody macromolecule technique using hemocyanin for the identification of type B and type C retrovirus envelope and cell surface antigens by correlative fluorescence, transmission electron, and scanning electron microscopy.
Authors: Gonda M A , Gilden R V , Hsu K C
Published: April 2011

The present resolution (75-100 A) of the conventional scanning electron microscope (SEM) and its ability to image the surfaces of large numbers of whole cells in situ permit the approach of problems such as viral and cell surface antigen localization by immunological labeling with visual markers. Identification of virus and cell surface antigens in situ has been accomplished in indirect reactions by unconjugated markers. Hemocyanin (Hcy) from whelk, Busycon canniculatum, has been developed as an immunospecific marker for virion and cell surface labeling in the electron microscope. Its size (30 x 50 nm) and distinct cylindrical shape permit easy visualization in the SEM and the transmission electron microscope (TEM). The Hcy method involves the preparation of antisera to Hcy in appropriate hosts for use in an unlabeled antibody macromolecule procedure based exclusively on antigen-antibody affinity to couple the macromolecule to the antigen site. Further correlative data from fluorescence microscopy can be obtained from similarly labeled samples by binding fluorescein to the bridging antibodies used in the Hcy technique. The usefulness of the Hcy marker system was demonstrated by employing highly specific antisera to the major envelope and cell surface glycoprotein (gp70) of Rauscher murine leukemia virus (R-MuLV), a type C retrovirus. The antiserum was shown to bind to the virion and cell surfaces of virus-infected cells in the homologous virus-infected cell system. It also demonstrated the expression of R-MuLV gp70-related antigens on a murine cell line Mm5mt/c1 which produces mouse mammary tumor virus (MuMTV), a type B retrovirus. Furthermore, when used in the Hcy marker system the anti-gp70 serum was able to distinguish type B from type C budding virus on the same cell. Methods for the preparation of immunoreagents and labeling of cells are discussed.
Elevated proteinase activities in mouse lung tumors quantitated by synthetic fluorogenic substrates.
Authors: Grabske R , Azevedo A , Smith R E
Published: April 2011

Proteinase activities in malignant and normal lung tissues were measured using two synthetic substrates that consist of a fluorophor coupled to a peptide moiety. The hydrolysis of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide and BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide were studied in homogenates of two types of mouse lung tumors, the Lewis lung tumor of the C57 black mouse and the KHT tumor of the C3H mouse. The activity of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.3 and a Km of 2.1 x 10(-4) M, required a thiol activator, and was inhibited by leupeptin suggesting the activity of a cathepsin B-like enzyme. The activity of BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.7 and a Km of 3 x 10(-5) M. Lung tumor homogenates contained higher hydrolytic activities for both substrates than normal lung homogenates.
Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells.
Authors: Sannes P L , McDonald J K , Allen R C , Spicer S S
Published: April 2011

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.
Quantitation and resolution in electron microscope radioautography.
Authors: Nadler N J
Published: April 2011

Electron microscopy can resolve structures with accuracy of the order of 0.2-0.3 nm, but the radioautographic technique is able to locate radioactive label with much less certainty, at best 50 nm. This difference in resolution is what creates the problem of quantitation in electron microscope radioautographs.
Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.
Authors: Hansen B L , Hansen G N , Vestergaard B F
Published: April 2011

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.
Evaluation of the validity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease.
Authors: Taper H S
Published: April 2011

The results of several tests and the characteristic morphological distribution of the enzymatic activity appeared to be in favor of the validity and specificity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease (DNAse) detection. These tests included thermal inhibition, omission of substrate, use of different chemical inhibitors and the reproduction of histochemical staining on Coujard's slides. Most of these results were in conformity with the biochemical data gathered from literature. Topographically selective inhibition of alkaline or acid DNAse by different factors suggested that there might exist two kinds of alkaline or acid DNase--one cytoplasmic and the other one nuclear. The whole histochemical procedure produced relatively small loss of alkaline and acid DNAse activities as verified by biochemical methods.
Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.
Authors: Vorbrodt A , Borun T W
Published: April 2011

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.
Dynamic assay of enzyme activities in single cells by flow cytometry.
Authors: Dolbeare F
Published: April 2011

Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.
A statistical evaluation of the binding of ferritin-conjugated lectins to the surface of rat cells. Topographical variations as a function of lectin concentration and cell type.
Authors: Hixson D C , Miller M F , Maruyama K , Walborg E F , Wagner S , Starling J J , Bowen J M
Published: April 2011

Statistical procedures were utilized to evaluate the concentration dependence of labeling by ferritin-conjugated lectins on four different rat cells: hepatocytes, normal thymocytes, Friend virus-induced rat tumor cells and feline sarcoma virus-induced rat sarcoma cells. Labeling by ferritin conjugates of concanavalin A, wheat germ agglutinin and Ricinus communis agglutinins I and II was quantitated by counting the number of ferritin granules on 600 Angstrom membrane segments. Relationships between the arithmetic means and variances for sample populations from each cell and ferritin-lectin combination were used to define four types of topographical distributions: uniform/ordered, uniform/random, random and clustered. It was found that the distribution and/or density of surface-bound lectin was concentration-dependent for all four ferritin-lectins. The nature of this dependency was complex and varied with both lectin and cell type.
Podocytes in aminonucleoside glomerulonephritis investigated ultrastructurally with concanavalin A.
Authors: Bretton R , Rouchon M , Bariety J
Published: April 2011

Peroxidase labeled concanavalin A (Con A) permits the detection of some saccharide determinants. This histochemical technique permits the visualization of cellular pathological modifications not observed with other methods. Its use in an ultrastructural study of nephritis induced by a single injection of aminonucleoside demonstrated the following in podocytes. The Con A positive endoplasmic reticulum (ER), essentially the rough ER, lost its normal linear and network appearance to take on a dot dash pattern. ER contents but not attached ribosomes and membranes were Con A positive. The dot dash pattern, due to a fragmentation of the ER, appeared prior to the onset of proteinuria and was attenuated before the disappearance of proteinuria. These changes of the ER were not observed in other proteinuric states. This suggests that aminonucleoside can damage the synthesis apparatus of podocytes, revealed by the Con A method.
The development of the sinusoids of fetal rat liver: localization of endogenous peroxidase in fetal Kupffer cells.
Authors: Pino R M , Bankston P W
Published: April 2011

Endogenous peroxidase is the cytochemical marker used to identify Kupffer cells in the adult liver. In this study, we show by ultrastructural cytochemistry that Kupffer cells of the fetal rat liver are endogenous peroxidase positive. The reaction product is localized in the endoplasmic reticulum including the perinuclear cisternae and in a few lysosome-like dense bodies. Serial sections of Golgi regions suggest that GERL and not the Golgi stacks, is peroxidase positive. As in the adult liver, peroxidase is not localized in endothelial cells. Kupffer cells do not appear to transform from endothelial or extravascular developing monocytic cells and are present prior to bone marrow formation. The relevance of these observations with respect to the possible origin of the Kupffer cell is discussed.
Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.
Authors: Williams D M , Gillett R , Linder J E
Published: April 2011

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.
The use of the potassium pyroantimonate-osmium method as a means of identifying and localizing calcium at the ultrastructural level in the cells of calcifying systems.
Authors: Appleton J , Morris D C
Published: April 2011

Potassium pyroantimonate-osmium has been used to localize calcium as an electron dense precipitate in the odontoblast, ameloblast and early hypertrophic chondrocytes of the mandibular condylar growth cartilage. The precipitate exhibited a specific association with various cell organelles. The presence of calcium in the precipitates was confirmed using energy dispersive analysis by x-rays. It is suggested that this K-pyroantimonate method provides an accurate technique for the investigation of the subcellular localization of calcium in calcifying systems.
Staining properties of aldehyde fuchsin analogs.
Authors: Buehner T S , Nettleton G S , Longley J B
Published: April 2011

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.
Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.
Authors: Le Bouteiller P P , Toullet F , Righenzi S , Voisin G A
Published: April 2011

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.
Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity.
Authors: Douglas W H , Hitchcock K R
Published: April 2011

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.
Tissue fixation by osmium tetroxide. A possible role for proteins.
Authors: Nielson A J , Griffith W P
Published: April 2011

The osmiophilia, under the conditions of normal tissue fixation, of the histidine, lysine, tryptophan, cysteine and methionine side chain of proteins is suggested by in vitro studies on blocked amino acids representative of such protein side chains, and the chemical nature of the reaction products elucidated. The chemical feasibility of inter- or intramolecular cross-linking of protein by OsO4 at these and other sites is demonstrated, as in the cross-linking of protein with unsaturated lipids such as methyl oleate, methyl linoleate and linolenate, and cholesteryl acetate. The relevance of these results to the process of tissue fixation by OsO4 is discussed.
Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells.
Authors: Willingham M C , Yamada S S
Published: April 2011

We have developed a new primary fixative that permits the localization of intracellular antigens with well preserved ultrastructural morphology. This primary fixation method employs a mixture of a water soluble carbodiimide with glutaraldehyde, and preserves morphology, yet produces a permeable cytosol matrix so that antibodies can gain access to fixed proteins. Cultured cells were primarily fixed, treated with detergent to permeabilize their membranes, reacted with peroxidase labeled antibodies, secondarily fixed, and embedded in situ. The variations in morphology and accessibility of intracellular antigens were evaluated for a variety of fixatives. Concanavalin A and alpha 2 macroglobulin were chosen as examples of intracellular protein antigens to evaluate these fixation methods. Both of the proteins were localized in intracellular vesicles.
Immunofluorescent localization of 25-hydroxyvitamin-D3-1 alpha-hydroxylase ferredoxin in the renal tissue of chick.
Authors: Ghazarian J G , Garancis J C
Published: April 2011

The chick renal mitochondrial 25-hydroxyvitamin-D3-1 alpha-hydroxylase is composed of three proteins, namely, cytochrome P-450, iron-sulfur protein (ferredoxin) and flavoprotein. Antibodies were raised in rabbits against homogeneous preparations of the ferredoxin. The antibodies were used in indirect immunofluorescence studies to localize the ferrdoxin along the nephron of renal tissues obtained either from vitamin D3-deficient or vitamin D3-sufficient chicks. The ferredoxin is predominantly localized in the glomerulus and proximal convoluted tubules. These results suggest that, in addition to the mitochondrial localization of the 1-hydroxylase, the enzyme may also be present in renal nuclei. The amount of the ferredoxin in kidney, as evidenced by the intensity of fluorescence, appeared to be independent of the vitamin D status of the chick. This finding indicated that changes in the concentration of the renal ferredoxin is not a major factor in the regulation of the 1-hydroxylase activity.
Spectra of cells in flow cytometry using a vidicon detector.
Authors: Wade C G , Rhyne R H , Woodruff W H , Bloch D P , Bartholomew J C
Published: April 2011

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.
Electronic sorting of granulocyte precursor cells from stimulated bone marrow.
Authors: Grogan W M , Scott R B , Collins J M
Published: April 2011

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.
Intracellular labeling with ferritin conjugates. A specificity problem due to the affinity of unconjugated ferritin for selected intracellular sites.
Authors: Parr E L
Published: April 2011

Nonspecific binding of ferritin to chromatin and the cytoplasmic aspect of the nuclear envelope was observed when nonantigenic, serum-washed hepatocyte nuclei were incubated in ferritin-antibody conjugates. This labeling was duplicated when nuclei from a wide range of species and cell types were exposed to unconjugated ferritin. Unconjugated ferritin binding to nuclei did not depend on a subpopulation of denatured molecules or on the ferritin purification procedure. Binding occurred equally on unfixed and formaldehyde-fixed nuclei, but no ferritin bound to glutaraldehyde-fixed nuclei. Inconjugated ferritin also bound to the cytoplasmic aspects of the rough endoplasmic reticulum and the plasma membrane. The tracer did not bind to lysosomes, mitochondria, Golgi vesicles, the extracellular surface of plasma membranes, or the intracisternal surfaces of ruptured nuclear envelopes. The addition of 0.4 M KCl or 0.7 M NaCl to ferritin solutions and washing media at neutral pH reduced the binding of conjugated and unconjugated ferritin to nuclei to about 3% of that seen in 0.10 M phosphate buffer alone. The added salts caused little extraction of nuclear contents from formaldehyde-fixed nuclei. The use of one of these salts in ferritin conjugates should considerably improve the specificity of intracellular labeling.
The demonstration of antibody specificity by a new technique. The gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) and its application to antibodies specific for feline leukemia virus.
Authors: Lutz H , Higgins J , Pedersen N C , Theilen G H
Published: April 2011

By combining the high resolution of sodium dodecylsulfate polyacrylamide gel electrophoresis with the sensitivity of enzyme-linked immunosorbent assay (ELISA) antibodies specific for different feline leukemia virus components are characterized. Based on the same principle, Concanavalin A binding sites of FeLV components are also detected.
Considerations for establishing the validity of immunocytological studies.
Authors: Hutson J C , Childs G V , Gardner P J
Published: April 2011

Immunocytology has wide spread applications for localizing tissue antigens, as evidenced by the recent exploitation of this technique in biological studies. Documenting the immunological specificity of the staining reaction is one of the most important technical considerations in validating the accrued data in immunocytological studies. The purpose of this report is to discuss and emphasize the need for conducting physiological studies in addition to the traditional immunological method and specificity controls. The ability of antibodies to bind molecules other than those molecules used as the immunizing material is a well documented fact. Hypothetically, preabsorption of the primary antibody with its specific antigen, could reduce subsequent binding of this antibody to a cross reactive tissue antigen, thus providing false confirmation of staining validity. The results of our experience with a cross reacting system in addition to other previously reported examples are discussed.
Dopamine receptor sites in the anterior pituitary.
Authors: Goldsmith P C , Cronin M J , Weiner R I
Published: April 2011

An immunocytochemical method was developed to visualize dopamine receptor sites on dispersed anterior pituitary cells of the rat. Dopamine receptors were labeled with the antagonist haloperidol. Some cells were incubated with haloperidol and a 100-fold excess of the potent antagonist D-butaclamol to determine nonspecific binding. The labeled sites were stained with an antibody against haloperidol and the peroxidase anti-peroxidase (PAP) technique. PAP complexes which served as markers for dopamine binding sites appeared on the outer plasmalemmal surface of the vast majority of mammotrophs. PAP complexes attached to the inner surface of endocytotic vesicle membrane suggested internalization of receptor-rich portions of the plasmalemma. Some gonadotrophs and somatotrophs were specifically stained to a lesser extent. However, high receptor site density and internalization of PAP complexes were never observed on cell types other than mammotrophs. The presence of dopamine receptors on the plasmalemma of mammotrophs provides strong additional evidence that dopamine acts upon these cells as a prolactin inhibitory hormone.
The use of avidin-biotin interaction in immunoenzymatic techniques.
Authors: Guesdon J L , Ternynck T , Avrameas S
Published: April 2011

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.
Complement-mediated binding of immunoglobulins to some endocrine cells of the pancreas and gut.
Authors: Buffa R , Solcia E , Fiocca R , Crivelli O , Pera A
Published: April 2011

Unspecific binding of immunoglobulins to gastrin, somatostain and glucagon cells of the gastrointestinal mucosa or pancreas has been found to occur through a nonantigen-antibody mechanism mediated by the C14 fraction of complement. The phenomenon represents an important drawback in hormone immunohistochemistry, which can be overcome by using complement deprived, highly dilute anti-hormone sera.
Innervation of the pancreas by substance P, enkephalin, vasoactive intestinal polypeptide and gastrin/CCK immunoractive nerves.
Authors: Larsson L I
Published: April 2011

Immunocytochemical studies habe shown that many peptides which profoundly affect the endocrine and exocrine functions of the pancreas are localized to neurons. In the cat, such peptidergic nerves appear to innervate ganglia, islets and blood vessels of the pancreas, whereas their contributions to exocrine cells are minor. Our studies suggest that pancreatic ganglia represent one major site of action of the peptides and that, in addition, nerves containing the vasoactive intestinal polypeptide and gastrin/CCK-related peptides profoundly affect pancreatic blood flow and insulin secretion, respectively.
Ultrastructural localization of neurophysin-like immunoreactivity in the rat posterior pituitary. An alternative method using frozen-dried tissue fixed with OsO4 vapor.
Authors: Coulter H D , Elde R P
Published: April 2011

Electron microscopic identification of elements containing neurophysin-like immunoreactivity can be accomplished in rat posterior pituitary that has been frozen-dried and fixed with OsO4 vapor. Alternating serial ultrathin sections are placed on grids and glass slides. The sections on the slides are stained for neurophysin using immunofluorescence histochemistry, and the resultant images are superimposed on electron micrographs from the adjacent sections. The method provides several advantages for the localization of neuropeptide immunoreactivity in nervous tissue.
Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342.
Authors: Loken M R
Published: April 2011

Data are presented that show that a histochemical stain, Hoeschst 33342, can be used to discriminate between viable B and T lymphocytes in the mouse. Quantitative analysis of the staining of cells from various lymphoid tissues with Hoechst 33342 using a Fluorescence-Activated Cell Sorter (FACS) indicates that two populations of cells can be identified. In the spleen approximately 60% of the lymphocytes can be classified as brightly stained with 1 microgram/ml of Hoechst 33342, while in the lymph node only 40% of the cells stain brightly. Thymocytes exhibit only the dull staining profile. Separation of these two populations from the spleen using the FACS and reanalyzing them for cell surface antigenic markers shows that the lymphocytes stained brightly with Hoechst 33342 are predominantly immunoglobulin positive, while the cells that stain less brightly express Thy 1.2. This indicates that a histochemical stain correlates directly with classical immunological markers on cell surfaces.
Problems inherent in obtaining the alkaline phosphatase reaction.
Authors: Doty S B
Published: April 2011

Problems encountered in the histochemical localization of alkaline phosphatase activity are discussed and solutions presented. The purpose is to achieve a reaction that can be studied by light and electron microscopy and to distinguish alkaline glycerophosphatase from inorganic pyrophosphatase activity. Details are presented concerning fixatives, fixation times, incubation media, enzyme inhibitors, activators, and associated techniques that can be used to obtain optimal histochemical results.
Sensitivity in electron microscope autoradiography for calcium-45.
Authors: Harris W V , Salpeter M M
Published: April 2011

The sensitivity of Ilford L4 emulsion to 45Ca was determined for electron microscope autoradiographic conditions. Sensitivity values were obtained for monolayers and double layers of emulsion in combination with various developing procedures. The dependence of sensitivity upon radiation dose was determined. All results are compared to previously calibrated isotopes.
A rapid method of cryofixation of tissues in situ for ultracryomicrotomy.
Authors: Chang S H , Mergner W J , Pendergrass R E , Bulger R E , Berezesky I K , Trump B F
Published: April 2011

A device is described for the rapid freezing of tissue in situ by a punch biopsy approach using a specially designed cryogun with a highly thermal conductive specimen holder. The cryogun consists of a sampling device using a double, spring-loaded gun mechanism and a system of cryochambers. Ultrathin freeze-dried sections cut from samples obtained with this cryogun are relatively free of artifacts and have few ice crystals. Organelles are seen by natural contrast when cryosections of approximately 1000 A are observed with a transmission electron microscope or in the transmission mode of a scanning electron microscope. The construction of the cryogun is described along with a method of obtaining improved, ultrafast cryofixation of tissue specimens. The reliability of obtaining x-ray microanalysis measurements of diffusible ions where movement within cell compartments has been retained is discussed.
Effects of different fuchsin analogs on the Feulgen reaction.
Authors: Teichman J S , Krick T P , Nettleton G S
Published: April 2011

The Feulgen reaction is used for cytophotometric quantitation of nuclear DNA. Schiff's reagents used in the Feulgen reaction usually are prepared from basic fuchsin, a variable mixture of four triaminotriphenylmethane analogs. The effect of the several fuchsin analogs on the quality of Schiff's staining of hydrolyzed DNA is not known. In this investigation Schiff's reagents prepared from relatively pure fuchsin analogs were used to determine whether different fuchsin analogs affect the absorbance of the Schiff's reagent-DNA complexes formed in solution. It has been determined that the complex formed by pararosaniline-Schiff's reagent and hydrolyzed DNA exhibits lower absorption than do corresponding complexes formed by Schiff's reagents prepared from magenta II or from new fuchsin.
Flow microfluorometric analysis of cellular DNA: Critical comparison of mithramycin and propidium iodide.
Authors: Hamilton V T , Habbersett M C , Herman C J
Published: April 2011

Mithramycin and propidium iodide were used to stain HeLa cells, human lymphoma cells, and phytohemagglutinin-stimulated lymphocytes for flow microfluorometric analysis of cellular DNA. The stains provided similar estimates for the proliferative fraction of the populations. However, significant differences in the relative fluorescent intensity were demonstrated in the three cell populations. Fluorescent intensity of HeLa and lymphoma cells stained with mithramycin was higher than matched propidium iodide-stained cells. Normal lymphocytes showed greater fluorescent intensity when stained with propidium iodide. Differences in the staining behavior of these two dyes may prove to be highly informative probes of chromatin structural differences.
Cytochemical localization of arylsulfatase B in rat basophils and mast cells.
Authors: Bentfeld-Barker M E , Bainton D F
Published: April 2011

Basophils and mast cells possess large metachromatically staining granules which contain sulfated glycosaminoglycans as well as vasoactive compounds. To determine whether these granules might also have lysosomal properties, we used electron microscopy and cytochemistry to localize arylsulfatase B in rat basophils and mast cells. In basophils of bone marrow, enzymatic reaction product was consistently seen in many, but not all, of the basophil granules. In some cells, the enzyme could also be demonstrated in the Golgi region, restricted to a single cisterna and small vesicles. It was never seen in rough endoplasmic reticulum (RER), although the paucity of cells made adequate sampling difficult. In mast cells of bone marrow and the peritoneal cavity, enzymatic reaction product was consistently found in some cytoplasmic granules of varying sizes and shapes where it characteristically rimmed the periphery of the granule just beneath the limiting membrane. It should be emphasized, however, that the majority of granules were not reactive. Reaction product could also be found occasionally in segments of RER, and in the Golgi region with a distribution similar to that of the basophil. The presence of lysosomal arylsulfatase in granules of developing basophils in bone marrow suggests that some basophil granules, like those of neutrophils, eosinophils, and monocytes are primary lysosomes. Some mast cell granules also contain this lysosomal enzyme, although it is not clear from the present data whether these granules are primary or secondary lysosomes.
Estrogen receptor cytochemistry by fluorescent estrogen.
Authors: Nenci I , Dandliker W B , Meyers C Y , Marchetti E , Marzola A , Fabris G
Published: April 2011

A recently synthesized fluorescein-labeled estrogen (17FE, 1-(N)-fluoresceinyl-estrone-thiosemicarbazone) interacts with estrogen-target cells like the native hormone and visualizes the uptake, transport, and distribution of estrogen in intact target cells. Moreover, estrogen binding sites are traced by 17FE in cryostat sections of estrogen target tissues as well. Cell and tissue 17FE binding sites fulfill the accepted criteria for specific estrogen receptors (finite binding capacity, high affinity, steroid and tissue specificity). This fluorescent probe allows estrogen receptors to be studied in a wide variety of cell and tissue preparations under varying conditions of physiologic and pathophysiologic interest.
Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.
Authors: Wang Y L , Taylor D L
Published: April 2011

The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein (5-IAF). The conjugate is purified by DEAE-cellulose ion exchange chromatography and cycles of polymerization-depolymerization, yielding a relatively homogeneous product with the fluorescein group covalently attached to cystein 373. The fluorescently labeled actin maintains normal polymerizability and activates heavy meromyosin Mg2+ adenosine triphosphatase to the same extent as unlabeled actin. Furthermore, fluoresecent paracrystals are readily detectable in fluroescence microscope upon adding excess Mg2+ or Ni2+ ions. Spectrofluorimetric studies of the bound fluorescein indicate that the peak excitation and emission wavelengths, the shapes of the spectra, and the peak fluorescence intensities are somewhat sensitive to polymerization and heavy meromyosin binding. Possible causes of these spectral changes are analyzed and future applications of this fluorescently labeled actin in vitro as well as in vivo are discussed.
X-ray microanalysis of aldehyde-fixed glycogen contrast-stained by OsVI . FeII and OsVI . RuIV complexes.
Authors: de Bruijn W C , van Buitenen J M
Published: April 2011

The composition of the contrast-donating complex of rat liver glycogen, nucleoplasm, erythrocytes, and mitochondria was established by X-ray microanalysis. In these compartments the presence of osmium and iron was shown qualitatively in tissue after glutaraldehyde fixation, treated with OsVIIIO4 plus K4FeII(CN)6 and in similar tissue treated with a combination of K2OsVIO4 plus K4FeII(CN)6. Osmium and ruthenium were detected in these compartments, in aldehyde-fixed tissue treated with mixtures containing K2RuIVL(CN)6 rather than K4FeII(CN)6. The iron detected in the glycogen, nucleoplasm, erythrocytes, and mitochondria of tissue treated with K2RuIV(CN)6 mixtures proved to derive from sources inside the electron microscope, and had to be considered an artifact. Quantitatively, the mean atomic ratios of osmium-to-iron and osmium-to-ruthenium were determined from spectra obtained by point analyses of the same compartments (glycogen, nucleoplasm, mitochondria, lipid droplets, and erythrocytes). After correction of the spectra for the instrumental iron contribution, the osmium-to-iron and osmium-to-ruthenium ratios in the glycogen were about 1:3 for tissue treated with those combinations including K2OsVIO4. In the other compartments, the osmium-to-iron and osmium-to-ruthenium ratios were virtually 1:0. For Os-VIIIO4 in combination with potassium ferrouscyanide however the osmium-to-iron ratio was 1:7 in the glycogen and 1:5 in all other compartments. OsVIIIO4 was combined with potassium ruthenium-cyanide, the osmium-to-ruthenium ratio was 1:2 in the glycogen and 2:1 in the other compartments. These results support our view that the selective glycogen contrast is obtained by complex formation.
Identification of contractile proteins in basal bodies of ciliated tracheal epithelial cells.
Authors: Gordon R E , Lane B P , Miller F
Published: April 2011

To determine the molecular composition of the components of basal bodies and the interbasal body apparatus of ciliated cells in rat tracheal epithelium, we used rabbit anti-actin, anti-alpha-actinin, anti-tropomyosin, and anti-myosin as primary antisera applied to the tissue in an indirect immunoperoxidase technique. The antisera was proven to be monospecific by elution of antibody after affinity chromatography. Sheep anti-rabbit immunoglobulin Fab fragments coupled to peroxidase were used for ultrastructural localization of the bound rabbit antibody. Antibodies against alpha-actinin were demonstrated around peripheral microtubules of cilia and linking these microtubules to central doublet and plasma membrane. Alpha-actinin was also shown in the basal foot processes. Anti-actin antibodies were associated with microtubules of the cilium and basal bodies, except in the region of the ciliary necklace. The antibodies directed against actin also had affinity for rootlets, basal foot processes, and communications between basal bodies and foot processes. Both anti-myosin and anti-tropomyosin antibodies were localized to part of the region of the constriction of the cilium, to the central basal density and the outer surfaces of basal body microtubules, and to the basal foot processes together with their communications to the basal body. The data suggest active contractile function of basal bodies.
A neurochemical and immunocytochemical study of P2 protein in human and bovine nervous systems.
Authors: DeArmond S J , Deibler G E , Bacon M , Kies M W , Eng L F
Published: April 2011

The anatomical distribution of P2 protein was studied in human autopsy tissue. Spinal cord (SC) and peripheral nerve (PN) were stained by the peroxidase-antiperoxidase method with antisera to bovine P2, glial fibrillary acidic protein, and myelin basic protein (BP). P2 antiserum did not stain all of the myelin in the PN. The staining was randomly distributed and discontinuous along a given myelinated axon. P2 antiserum also stained SC myelin in a pattern similar to the PN. Only a fraction of the sheaths stained, in contrast to BP antiserum that stained all myelin sheaths in both the SC and PN. P2-positive myelin was distributed throughout the SC white matter, including an occasional myelinated fiber in the SC grey matter. P2 and BP antisera did not stain regions of demyelination in a case of idiopathic polyneuritis, while adjacent myelinated PN stained normally. Absorption of the P2 antiserum with P2, bovine PN or bovine SC (carefully dissected to eliminate PN contamination) nullified the specific staining in both the PN and SC; however, absorption with BP or hemispheric myelin did not eliminate P2 staining. The P2 antiserum formed a single immunodiffusion line with pure P2 and acid extracts of bovine SC and PN myelin, but not with an acid extract of bovine hemispheric myelin. Electrophoresis of defatted bovine SC produced a distinct band corresponding to P2. Therefore, three lines of evidence, immunocytochemical, immunodiffusion and electrophoretic, suggest that P2 is present in PN and SC but not in hemispheric myelin.
Immunoenzymatic localization of prolactin-like immunoreactivity in decidual cells of the endometrium from pregnant and nonpregnant women.
Authors: Meuris S , Soumenkoff G , Malengreau A , Robyn C
Published: April 2011

Prolactin-like immunoreactivity has been localized by an immunoenzymatic method in the decidual cells of the endometrium in cases of early (10 weeks) normal pregnancy, (12 weeks) molar pregnancy, (8--10 weeks) tubal pregnancy, and of a nonpregnant woman under progestogen (lynestrenol) treatment. A specific endocrine influence, predominantly progestogenic, rather than the implantation of trophoblastic tissue seems to be required for the appearance of prolactin-like immunoreactivity in the decidual cells of the endometrium.
An attempt at quantitation of procollagens I and III and of collagen IV in tooth sections by exposure to the corresponding antibodies followed by 125I-protein A and radioautography.
Authors: Wright G M , Leblond C P
Published: April 2011

The immunoreactivity of procollagen types I and III and of collagen type IV was detected in frozen sections of the growing apical end of rat incisor teeth by an indirect method making use of protein A. The sections were exposed to affinity-purified antibodies against these substances. The bound antibodies were then detected by incubation with radioiodinated protein A, followed by radioautography. This immunoradioautographic approach yielded preparations with low background, in which the reactions could be quantitated by counts of silver grains. The distribution of the radioautographic reactions was essentially the same as that previously observed with direct and indirect peroxidase methods, that is, procollagen I antigenicity predominated in odontoblasts and predentin, with minor amounts in periodontal tissue and pulp; procollagen III antigenicity was present in periodontal tissue and, to a lesser extent, in the pulp; and collagen IV antigenicity was restricted to basement membranes. Moreover, grain counts provided quantitative support for the conclusions on the distribution of procollagen I and III antigenicity.