bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin complex binding to human peripheral and mitogen-stimulated lymphocytes.
Authors: Cunningham V L , Wolken K W , Ackerman G A
Published: April 2011

Surface labeling and internalization of insulin was demonstrated ultrastructurally with human peripheral lymphocytes and with "activated"/transformed lymphocytes from mitogen-treated cultures using the colloidal gold-labeled insulin-bovine serum albumin (GIA) procedure. The majority of peripheral lymphocytes bound only limited amounts of the insulin complex, while approximately 15% of the lymphocyte population bound modest to comparatively large quantities of the labeled hormone. Quantitative labeling data indicated a skewed GIA labeling continuum for peripheral lymphocytes rather than separate, distinct populations. Sequential labeling studies with the GIA complex followed by either the ferritin-conjugated goat anti-human immunoglobulin or the E-rosette techniques indicated that insulin labeling was neither T nor B cell specific, since extremes of GIA labeling were found in both populations. Many, but not all, circulating lymphocytes with elevated insulin binding had morphological features suggestive of "active" cells, viz., larger cell, nuclear, nucleolar, and Golgi sizes, dispersed chromatin, and greater numbers of polysomes than lymphocytes having minimal GIA labeling. Both phytohemagglutinin (PHA), a T-cell mitogen, and pokeweed mitogen (PWM), a B/T cell mitogen, induced an increase in mean GIA labeling of cultured lymphoid cells as compared to non-mitogen-treated controls. The majority of mitogen-transformed "blast-like" cells had more extensive insulin labeling than nontransformed small (medium)-size lymphocytes, although an overlap in labeling densities was noted in these two groups. PHA induced a slight increase in mean surface GIA labeling of the nontransformed lymphocyte population at 48 and 72 hr of culture as compared to similar cells in non-mitogen-treated controls and PWM cultures. We interpret these findings as indicating the emergence of increased numbers of insulin binding sites on lymphocytes, both those in the circulation and in mitogen-treated cultures, during the early response (activation) to functional and/or metabolic modulations of the cell; this surface change does not appear to be directly related to blastogenic transformation.
High concentrations of a novel peptide, neuropeptide Y, in the innervation of mouse and rat heart.
Authors: Gu J , Polak J M , Allen J M , Huang W M , Sheppard M N , Tatemoto K , Bloom S R
Published: April 2011

A newly discovered bioactive peptide, neuropeptide Y (NPY), has been found in the innervation of the mouse and rat heart by immunocytochemistry, NPY-immuno-reactive nerves were very dense around the nodal tissues. They also surrounded the coronary arteries and arterioles and were found in close association with the cardiac muscle. The distribution of NPY-containing nerves paralleled that of noradrenergic fibers, demonstrated by the use of antibodies to the catecholamine-converting enzymes tyrosine hydroxylase and dopamine beta-hydroxylase. Furthermore, NPY was seen to be present in a proportion of intrinsic neurons mostly found in the atria and in close proximity to the nodal tissue. The concentrations of extractable NPY-immunoreactive material (about 150 pmol/g in whole mouse heart) by far surpasses those of the other peptides so far reported in the cardiac tissue. High performance liquid chromatography demonstrated the NPY immunoreactivity to elute in a single sharp peak, in an identical position to brain NPY.
Histochemical investigation of criteria for the distinction between monoamine oxidase A and B in various species.
Authors: Uchida E , Koelle G B
Published: April 2011

The superior cervical ganglion (SCG), pineal body (PB), and liver (L) of the rat, rabbit and cat were stained for monoamine oxidase (MAO) A and B by the tetranitro blue tetrazolium (TNBT) and coupled peroxidase ( PerOx ) methods, using 5-hydroxytryptamine (5HT), tryptamine ( Tryp ), tyramine (Tyr), and benzylamine (Bz) as substrates, and clorgyline (Cl) and deprenyl (Dep), both at 10(-7) M, as selective inhibitors. The nodose ganglion (NG) and dorsal root ganglion (DRG) of the rabbit and cat were also studied. The results with rat tissues were consistent with published quantitative findings (SCG, MAO-A much greater than B; PB, MAO-A less than or equal to B; L, MAO-A = B). In the rabbit, the findings with the SCG were similar; the MAO activities of the PB were relatively resistant to both inhibitors; the MAO of the liver required 10(-4) M concentrations of both inhibitors to produce near total inhibition, suggesting that the liver contains an MAO distinct from MAO A and B. All cat tissues examined appeared to contain almost exclusively MAO-B. In this species 5HT, which is generally considered a selective substrate for MAO-A, was oxidized by MAO-B. The findings indicate that criteria for MAO-A, -B, and other subgroups must be defined for each species and tissue.
Preembedding labeling with biotinylated antibodies and subsequent visualization of the biotin groups exposed on thin sections.
Authors: Nir I , Schneider B G , Papermaster D S
Published: April 2011

The feasibility of labeling cell membranes with biotinylated ligands and detecting the biotin groups on thin sections was investigated. Fixed retinal tissue was incubated with biotinyl- antiopsin . Half of the biotinyl-antibody labeled retinal tissue was incubated with avidin-ferritin (AvF) and embedded in Epon (preembedding reaction). The second half was embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Thin sections of this preparation were incubated with AvF to detect biotinyl-antibodies exposed by the sectioning (postembedding reaction). Biotin groups on the thin section surface could be readily visualized with AvF. Stereoscopic images demonstrated that the ferritin particles were localized only on the exposed surface of the thin section. The labeling was highly specific, with a very low background. Quantitative analysis was employed in order to determine the optimal reaction conditions for maximizing the labeling density with minimizing nonspecific binding. The possibility of using biotinylated molecules in the study of dynamic cellular events and for the subsequent intracellular localization of biotin on thin sections is suggested.
Peroxidase activity in human cutaneous mast cells: an ultrastructural demonstration.
Authors: Escribano L M , Gabriel L C , Sainz T , Rocamora A , Arrazola J M , Navarro J L
Published: April 2011

An intense and reproducible peroxidase staining in the cutaneous mast cells of two patients with systemic mast cell disease and urticaria pigmentosa is demonstrated at the ultrastructural level. This enzyme activity was demonstrated by use of a cytochemical technique employing 3,3'- diaminobenzicine (DAB) as an oxidizable substrate, after fixation by a tannic acid-aldehyde mixture. Enzyme activity was localized in the perinuclear cisterna and strands of endoplasmic reticulum. Granules appeared unreactive. This peroxidase activity appears sensitive to fixation by aldehydes; it is inhibited by 3-amino-1,2,4-triazole (AMT) and by lack of H2O2 or DAB in the incubation medium. These characteristics are fundamentally different from the peroxidase activity of basophils, and the demonstration of this enzyme is therefore not a further argument for a common ontogenetic origin of both cells. On the other hand, the cytochemical characteristics of this enzyme are very similar to those of platelet peroxidase (P-PO), which has been connected to the synthesis by platelets of prostaglandins. Since the mast cell is known to generate prostaglandins, the relationship between the enzyme described and prostaglandin synthesis by mast cells is discussed.
Lectin-binding histochemistry of non-decalcified growth plate cartilage: a postembedment method for light microscopy of epon-embedded tissue.
Authors: Farnum C E , Wilsman N J
Published: April 2011

A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.
Carbonic anhydrase in mouse salivary glands and saliva: a histochemical, immunohistochemical, and enzyme activity study.
Authors: Ikejima T , Ito S
Published: April 2011

Mouse parotid gland and saliva were studied by histochemical, immunohistochemical, and activity measurements for carbonic anhydrase. Hansson 's histochemical reaction for carbonic anhydrase revealed positive enzyme activity in the parotid acinar cell cytoplasm and little or no reaction in the secretory granules. The luminal contents in all of the glandular duct systems also reacted positively, but the duct cells themselves were only weakly positive. Ultrastructural observations confirmed the light microscope histochemical localization and, in addition, revealed luminal content activity in intercellular ducts. Purified carbonic anhydrase isolated from mouse salivary glands was used to raise antibodies in rabbits. Localization of carbonic anhydrase by direct immunolabeling with fluorescein-coupled antibody and indirect immunoperoxidase labeling revealed enzyme localization on or in the acinar cell secretory granule membrane and not in the surrounding cytoplasm. The luminal contents of the intercalated and striated ducts were also strongly positive. Stimulation of salivary secretion with phenylephrine or pilocarpine increased the amount of carbonic anhydrase in saliva. Acetatazolamide and potassium cyanate inhibited carbonic anhydrase activity. Reasons underlying the discrepancy between the histochemical and immunolabeling localization of carbonic anhydrase are discussed. It is concluded that the parotid acinar cells of mice appear to be a significant source of carbonic anhydrase in saliva but its role is enigmatic.
Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.
Authors: Scott C S , Hough D , Bynoe A G , Jones D B , Roberts B E
Published: April 2011

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.
Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody.
Authors: Glazer E J , Ramachandran J , Basbaum A I
Published: April 2011

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level.
The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.
Authors: Herbener G H , Bendayan M , Feldhoff R C
Published: April 2011

The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus. This labeling of lysosomes may be a consequence of the high blood plasma concentrations of vitellogenin that occur in the frog model, or to the well-known crinophagy phenomenon present in secretory cells.
Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.
Authors: Hisano S , Adachi T , Daikoku S
Published: April 2011

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.
Identification of calcium in the retina by the combined use of ultrastructural cytochemistry and laser microprobe mass analysis.
Authors: Van Reempts J L , Borgers M , De Nollin S R , Garrevoet T C , Jacob W A
Published: April 2011

The specificity of the combined oxalate-pyroantimonate (OPA) technique for subcellular localization of calcium was examined by laser microprobe mass analysis (LAMMA) of the same tissue sections. The recorded spectra strongly suggest that the cytochemically detected precipitates contained calcium. This could be confirmed by LAMMA analysis of ethylene glycol tetraacetic acid-treated sections. The detected calcium was localized mainly in the rod outer segments, more particularly in the middle part. The validity of the OPA and LAMMA methods is discussed. A combination of both techniques is found to be a valuable tool to elucidate the role of calcium in physiological mechanisms.
Models and concepts derived from human teratogenesis and oncogenesis in early life.
Authors: Bolande R P
Published: April 2011

Several basic empirical facts are emphasized about human developmental oncology. The first is that teratogenesis and oncogenesis are intimately related and that indeed teratogenesis may be the more primitive reaction to the types of mutagenic injury giving rise to neoplasm. The second is that neoplasms of early life, particularly those initiated "in utero" are rare, and tend to spontaneously regress or cytodifferentiate. The theoretical models of carcinogenesis forwarded by Knudson and Matsunaga are enlisted in attempts to explain these phenomena and how the oncogene is expressed and modulated by the fetal or embryonal milieu.
Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig.
Authors: Altschuler R A , Fex J , Parakkal M H , Eckenstein F
Published: April 2011

The guinea pig lateral superior olive was examined immunocytochemically using antisera against enkephalin and choline acetyltransferase sequentially on the same sections. A colocalization of choline acetyltransferase-like and enkephalin-like immunoreactivities was found in cells of the lateral superior olive that give rise to the lateral system of olivocochlear efferents. Only choline acetyltransferase-like immunoreactivity was observed in the group of olivary nuclei that give rise to the medial group of olivocochlear fibers.
Localization mechanisms in enzyme cytochemistry studied with alkaline phosphatase-loaded erythrocyte ghosts.
Authors: Raap A K , Van Duijn P
Published: April 2011

Erythrocyte ghosts containing varying amounts of alkaline phosphatase were used to study the localization mechanisms of three metal salt and one azo method for this enzyme. For the azo method, the minimal amount of alkaline phosphatase that can be visualized within the ghosts proved only to be limited by the optical properties of the azo compound. In contrast, for the metal salt methods, a certain threshold activity had to be present in the ghosts in order to obtain correct localization of the final reaction product. The localization properties of both azo and metal salt methods conformed to the theories of cytochemical enzyme localization presented to date. By determining the rate constant of the capture reaction and the diffusion constant of the primary product, the localization properties of the azo method could be predicted. Some remaining discrepancies between theory and practice are discussed.
Quantification of antiserum reactivity in immunocytochemistry. Two new methods for measuring peroxidase activity on antigen-coupled beads incubated according to an immunocytoperoxidase method.
Authors: Pool C W , Madlener S , Diegenbach P C , Sluiter A A , van der Sluis P
Published: April 2011

Antigens covalently coupled to agarose beads provide a matrix for an economical, sensitive, and quantitative immunocytochemical detection of antiserum bindings potencies. Despite some very powerful features (e.g., the ability to control the outcome of a solid phase adsorption on the same matrix), the use of this technique is not very widespread when compared with the other enzyme-linked immunosorbent assay (ELISA) techniques. The main reason for this is the necessity for rather laborious measurements of the immunocytochemical tracer on individual beads. A description of two new methods for the batch measurement of the peroxidase activity on immunoperoxidase incubated antigen-coupled beads is presented. The first method involves the measurement of the diaminobenzidine (DAB) extinction from a large number of beads with a scanning microspectrophotometer. In the second method, during the peroxidase reaction, the beads are incubated with o-phenyldiamine (OPD), which is soluble both in the reduced and oxidized form, whereby absorbance measurements of the supernatant of the beads in a normal spectrophotometer are possible. The sensitivity and the quantitative relation between bound first antibody and absorbance are compared for both methods. From the two immunoperoxidase procedures used (the three step peroxidase-antiperoxidase and the two-step peroxidase conjugate procedure) only the latter met the conditions for a quantitative (first) antibody assay.
The effect of Tween 20 on indirect immunoperoxidase staining of blood group antigen A in human urothelium.
Authors: Juhl B R , Nørgaard T , Bjerrum O J
Published: April 2011

The effect of the nonionic detergent Tween 20 on background staining, sensitivity, and specificity in the indirect immunoperoxidase staining for blood group antigen A was investigated histologically and spectrophotometrically. Pretreatment of dewaxed formalin-fixed Paraplast-embedded tissue sections from human ureters with 2% Tween 20 and dilution of the first and second layer antisera with 0.05 or 2% Tween 20 significantly reduced background staining of the urothelial cell cytoplasm, ureteral stroma, and musculature. Spectrophotometrical analysis of tissue sections from hypernephroma (rich in cytoplasm), cervix (fibrous stroma), and myometrium (musculature) underlined the histological results with a significant reduction of the maximum absorbance of Tween 20-modified indirect immunoperoxidase-stained tissue sections. Sensitivity, evaluated histologically by the endpoint titers of urothelial cell membrane staining, endothelial cell staining, and focal cytoplasmic staining of urothelial cells, was not influenced by the Tween 20 treatment. The specificity was improved as the staining was highly reduced or absent in control sections subjected to Tween 20.
Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates.
Authors: Laden S A , Schulte B A , Spicer S S
Published: April 2011

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.
Conjugated avidin binds to mast cell granules.
Authors: Tharp M D , Seelig L L , Tigelaar R E , Bergstresser P R
Published: April 2011

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.
An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.
Authors: Jones K H , Senft J A
Published: April 2011

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
Marginal zone macrophages in the mouse spleen identified by a monoclonal antibody. Anatomical correlation with a B cell subpopulation.
Authors: van Vliet E , Melis M , van Ewijk W
Published: April 2011

The reactivity of a monoclonal antibody, ER-TR9, that demonstrates heterogeneity among mononuclear phagocytes is described. In the spleen, ER-TR9 exclusively reacts with a population of macrophages located in the marginal zone. ER-TR9 does not react with macrophage antigen 1-positive red pulp macrophages or any other types of splenic stromal cells. ER-TR9+ ve cells localize in anatomical proximity of a subpopulation of B cells, i.e., B cells that are immunoglobulin M positive and weakly positive to negative for immunoglobulin D. The possible significance of this particular interaction between both cell types during the immune response is discussed.
Binding of Fab-horseradish peroxidase conjugates by charge and not by immunospecificity.
Authors: Pino R M
Published: April 2011

The influence of horseradish peroxidase (HRP) charge on Fab-HRP conjugates was investigated. Rabbit nonimmune Fab coupled via periodate or glutaraldehyde to Sigma HRP type VI (pI greater than 10) were cationic (positively charged) as determined by analytical isoelectric focusing. These conjugates and HRP type VI alone stippled the basal laminae and collagen fibers in Bruch's membrane of the rat eye in a pattern identical to anionic (negative) sites. Binding was not present after the anionic sites were removed by enzyme digestion prior to immunolabeling or when HRP type VIII (anionic with pI 3.6) was used in an Fab-HRP conjugate or in an unbound form. These results indicate that anionic HRPs should be used in Fab-HRP preparations if a nonspecific binding to anionic sites is possible.
Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization.
Authors: McAllister H A , Rock D L
Published: April 2011

Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.
Identification of gastrin-producing cells in cell cultures and smears: an avidin-biotin-peroxidase complex (ABC) technique.
Authors: Gower W R , McSweeney E M , Dyben T E , Fabri P J
Published: April 2011

A method is described that uses the avidin-biotin complex (ABC) immunoperoxidase technique to provide a rapid, sensitive, and specific means to quantitate isolated G cells in cultures and suspensions.
Intermediate filaments in the developing type II cell of fetal monkey lung.
Authors: Chi E Y , Gown A M , Vogel A M , Teh E C
Published: April 2011

Anticytokeratin monoclonal antibody was used to study epithelial cell development in fetal monkey lungs taken from animals of different ages. It is well established that the overall maturity of fetal lung depends greatly on the maturation of type II epithelial cells in the alveolus. In this study, we have correlated the cytokeratin phenotype of mammalian epithelial cells with pneumocyte maturation. We show that differentiation and maturation of the type II cell is related to intermediate filament expression. Twenty-four fetal monkeys (Macaca nemestrina) were delivered by cesarean section at a gestational age of 135-140 days (term = 168 days) and divided into two groups. One group of animals was sacrificed during the first 3 hr of life, and the other group was maintained in incubators for 92-120 hr. Anticytokeratin monoclonal antibody recognizes only alveolar type I and type II epithelial cells. In the first 3 hr of life, the cytokeratin was localized only at the alveolar surface and at the cytoplasmic periphery of the type II cells of these premature animals. However, at the age of 92-120 hr, the epithelia in the lungs reacted more intensely than they did during the first 3 hr. Electron microscopy revealed and confirmed that the type II cells were matured and abundant intermediate filaments appeared in the cytoplasm. The filaments appeared to form either aggregates or parallel filament bundles and few were closely associated with the lamellar bodies. In the immature type II cells at 0-3 hr of life, few intermediate filaments could be localized in the cytoplasm, and no parallel filament bundle was observed, though many appeared in the 92-120 hr lungs. This suggests that the intermediate filaments have a functional significance in the development and maturation of the type II cell. The location and stability of keratin filaments in type II cells may confer the structural strength necessary for cells covering a free surface in the alveoli during lung maturation.
A new fixative for the preservation of actin filaments: fixation of pure actin filament pellets.
Authors: Boyles J , Anderson L , Hutcherson P
Published: April 2011

Conventional fixation for thin-section microscopy is insufficient to preserve many elements of cells and tissues. Actin filaments, for example, are destroyed during post-fixation in OsO4. In our search for a better fixative, we chose pellets of pure actin filaments as a very sensitive model system. In the present study, the potential of amines for improving aldehyde fixation was explored, and the results were compared to those obtained with the use of tannic acid. Aldehyde and amine were used together as an initial fixative, followed by aldehyde alone with postfixation in 1% OsO4 in buffer at 4 degrees C for 15 min, uranyl acetate en bloc stain, acetone dehydration, and embedding in Epox 812. Some primary monoamines improved the preservation of filaments; filaments were not broken beyond recognition by OsO4, as occurs when glutaraldehyde alone is used. Excellent preservation was seen when certain primary diamines were used. The quality of this fixation was superior to that obtained with tannic acid and was without the large increase in filament diameter that is seen with concentrations of tannic acid sufficient to protect filaments against osmium damage. The effects on filaments of the amines lysine, putrescine, ammonium, and arginine have been documented in detail, as we systematically varied all the major parameters normally considered in formulating fixation protocols.
Three mouse monoclonal antibodies to human differentiation antigens: reactivity with two mucin-like antigens and with connective tissue fibers.
Authors: Mattes M J , Look K , Lewis J L , Old L J , Lloyd K O
Published: April 2011

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.
A modified histochemical technique to visualize acetylcholinesterase-containing axons.
Authors: Hedreen J C , Bacon S J , Price D L
Published: April 2011

An improved histochemical method for light microscopic demonstration of acetylcholinesterase (AChE) has been developed. Axonal, dendritic, and perikaryal staining are well delineated, both in areas of low AChE content, such as cerebral cortex, and in areas of high AChE content, such as neostriatum. Axonal staining, including arborizations, stands out against a clear background devoid of diffuse reaction product.
Topographic localization of a 116,000-dalton protein in cartilage.
Authors: Fife R S , Hook G L , Brandt K D
Published: April 2011

A disulfide-bonded greater than 400,000-dalton (greater than 400-kD) protein with 116-kD subunits in hyaline cartilage from several species has recently been described. It constitutes 2-4% of the total noncollagenous protein in 4 M guanidinium chloride extracts of normal articular cartilage and accounts for most of the total noncollagen, nonproteoglycan protein synthesized in short-term organ cultures of canine articular cartilage. In the present study, immunofluorescence techniques were used to examine the topographic distribution of the 116-kD subunit protein in normal cartilage. In specimens of normal adult articular cartilage from several species, the protein was located throughout the matrix. More intense staining was observed at the articular surface than in the remainder of the uncalcified cartilage. In contrast, in fetal cartilage, the protein was uniformly distributed throughout the matrix without a marked increase in surface staining. Normal canine menisci and annulus fibrosus also demonstrated moderate fluorescence after incubation with the antiserum to the 116-kD subunit protein. Normal canine nucleus pulposus, synovium, aorta, and monolayer cultures of canine synovial cells exhibited only weak immunofluorescence after incubation with the antiserum. Therefore, the 116-kD subunit protein appears to be a ubiquitous matrix protein in cartilage.
Immunohistochemical identification of the uncoupling protein in rat brown adipose tissue.
Authors: Cadrin M , Tolszczuk M , Guy J , Pelletier G , Freeman K B , Bukowiecki L J
Published: April 2011

Brown adipose tissue mitochondria are characterized by the presence of an uncoupling protein that gives them an exceptional capacity for substrate-controlled respiration and thermogenesis. The specific localization of this protein in rat brown adipocytes was demonstrated using an immunohistochemical technique, the peroxidase-antiperoxidase (PAP) method. Light microscopy observations showed that serum antibodies raised against the uncoupling protein selectively reacted with multilocular brown adipocytes. No labeling could be detected in either unilocular adipocytes, capillaries, or muscle fibers (striated and vascular smooth muscle). Staining was more intensive in certain adipocytes than in others, suggesting the presence of cellular heterogeneity. The specificity of the staining technique was demonstrated by showing that treatment of the preparations with antiserum saturated with an excess of uncoupling protein almost entirely inhibited brown adipocyte labeling. The specificity and selectivity of the PAP method allow the clear differentiation of uncoupling protein-containing adipocytes from other cellular types, suggesting that this immunohistochemical technique will represent an extremely useful tool for studying adipocyte function and differentiation.
Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: The influence of fixation on patterns of lectin binding.
Authors: Hardham A R
Published: April 2011

The study of the surface properties of zoospores and cysts of the fungus Phytophthora cinnamomi required a fixation regime that would preserve the cells adequately and not interfere with binding and detection of probes on the cell surface. When they were fixed in 4% formaldehyde (F), specific binding of concanavalin A-fluorescein isothiocyanate and rhodamine-labeled soybean agglutinin was obtained. However, electron microscopy showed that preservation was so poor that intracellular binding sites had become exposed. By contrast glutaraldehyde (G), even at concentrations as low as 0.05%, gave good preservation of the zoospores but induced high levels of nonspecific fluorescence, making its use impractical for studies using fluorescent probes. Addition of 1-4% F to 0.05-0.8% G reduced the level of G-induced fluorescence while not diminishing the quality of ultrastructural preservation. This effect was evaluated quantitatively and an optimum fixation regime for the fungal cells, namely, 0.2% G and 2-4% F in 50 mM PIPES buffer, was determined. This combined fixative facilities correlated fluorescence and ultrastructural labeling with lectins and immunocytochemical probes.
Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues.
Authors: Nathrath W B , Heidenkummer P , Björklund V , Björklund B
Published: April 2011

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.
Abscissic acid localization by light microscopic immunohistochemistry in Chenopodium polyspermum L. Effect of water stress.
Authors: Sotta B , Sossountzov L , Maldiney R , Sabbagh I , Tachon P , Miginiac E
Published: April 2011

An indirect immunohistochemical technique was developed using a rabbit anti-abscissic acid (ABA) serum and the soluble peroxidase-antiperoxidase (PAP) complex for the localization of endogenous ABA in the aerial parts of Chenopodium. Terminal bud, axillary bud bearing nodes, and adult leaves were prefixed by a soluble carbodiimide to obtain the coupling of ABA on cellular proteins and postfixed by a conventional mixture of aldehydes. They were then embedded in paraffin or in plastic. Numerous controls were carried out on sections and on a model system to test the validity of the technique. Based on the staining patterns observed along the plant, an apico-basal gradient of ABA was revealed. In the older buds, ABA was mainly concentrated in the quiescent meristematic cells of the apex. Phloem cells of the main axis and chloroplasts of the leaves were specifically labeled. No reaction product was visualized in the parenchyma cells or in the cambial zone. Water stress, which is known to increase ABA content, induced an increase of immunoreactivity within the same compartments. This physiological test validates the stain.
Quantitative electron probe x-ray microanalysis and densitometric mass determination of individual rat blood platelets.
Authors: Linders P W , Van de Vorstenbosch R A , Stadhouders A M
Published: April 2011

An improved method for quantitative electron probe X-ray microanalysis of thin biological specimens, introduced recently, has been applied to the elemental analysis of rat blood platelets. The method uses the X-ray signal to quantify the elemental content of an object and electron scattering to determine the total dry mass of the object. Along with the dry mass distribution, data were obtained on the content and mass fraction of calcium, magnesium, and sulfur in 31 individual platelets. The mean platelet dry mass was found to be 985 fg. The mean Ca, Mg, and S contents were 1.80, 3.41, and 18.3 fg, with mean dry mass fractions of 0.195, 0.396, and 1.96%, respectively. Furthermore, these elements appear to be unevenly distributed among the platelet population.
Multiple binding modes for Hoechst 33258 to DNA.
Authors: Stokke T , Steen H B
Published: April 2011

Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.