bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis.
Authors: von Bartheld C S , Cunningham D E , Rubel E W
Published: April 2011

The molecule 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is a fluorescent dye which diffuses within cell membranes. The properties of DiI diffusion and fluorescence are maintained in aldehyde-fixed tissue, thereby allowing selective neuronal tracing post mortem. We describe three modifications of this tracing method. First, while DiL diffuses along neuronal membranes the tissue can be decalcified in EDTA at 37 degrees C. Tracing in decalcified tissue extends the possible application of the DiI technique to the investigation of neuronal tissue enclosed in bony structures. Second, we describe a protocol that allows sectioning of DiI-injected tissue on a cryostat with minimal subsequent spread of DiI in dried sections. Third, we demonstrate that DiI label of fluorescent neurons in cryosections as well as Vibratome sections can be photo-oxidated and converted to a stable diaminobenzidine reaction product. The photo-converted DiI label is electron dense and allows analysis of labeled cell bodies and processes at the electron microscopic level. DiI does not stay confined to the surface cell membrane in fixed tissue but reaches internal organelles, presumably via membranes of the endoplasmic reticulum, and concentrates in microsomal structures adjacent to mitochondria. Photoconversion of DiI label is compatible with gold immunocytochemistry. Long-term incubation and subsequent photoconversion of post-mortem DiI-labeled neurons provides remarkable tissue preservation at the ultrastructural level.
High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.
Authors: Erlandsen S L , Bemrick W J , Schupp D E , Shields J M , Jarroll E L , Sauch J F , Pawley J B
Published: April 2011

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.
Quantitative immunohistochemistry of synaptophysin in human neocortex: an alternative method to estimate density of presynaptic terminals in paraffin sections.
Authors: Masliah E , Terry R D , Alford M , DeTeresa R
Published: April 2011

Currently available specific synaptic markers have made it possible to estimate the synaptic density by immunochemical techniques. In the present study we labeled the neocortical presynaptic terminals in histological sections of human autopsy tissue with a monoclonal antibody against synaptophysin. The characteristic granular neuropil reaction was quantified by measuring the average optical density (OD) in the different layers of the parietal cortex with the aid of image analysis equipment. The raw neuropil OD was corrected by subtracting the OD of the white matter in the same section. Our study showed that consistent microdensitometric results can be obtained on 5-microns paraffin sections from specimens with less than 8 hr of post-mortem time before fixation, incubated with 5 micrograms/ml of anti-synaptophysin. The corrected OD measurements were slightly larger in neocortical layers II, III, and V than in layers I, IV, and VI, but the differences were not statistically significant. In area 17, layer IV was denser than the others. We conclude that with certain precautions this method can be used to measure relative amounts of synaptophysin-like immunoreactivity and to infer the density of presynaptic boutons in human situations and in animal models.
Chromogranins A and B are co-localized with atrial natriuretic peptides in secretory granules of rat heart.
Authors: Steiner H J , Weiler R , Ludescher C , Schmid K W , Winkler H
Published: April 2011

We investigated the occurrence and subcellular localization of chromogranins A and B in atrial myoendocrine cells of rat heart, using immunological methods. Immunoblotting revealed the presence of both chromogranin A and B in an extract from large granules isolated from this tissue by subcellular fractionation. Immunohistochemistry at the ultrastructural level demonstrated the presence of chromogranin A and B in secretory granules. These organelles also immunostained for atrial natriuretic peptides (ANP). Within a given section, all granules were labeled with immunogold for these three antigens. This apparent co-localization of the three antigens was confirmed by double immunostaining with immunogold particles of different sizes. We conclude that, in agreement with their endocrine nature, the secretory organelles of rat atria contain both chromogranins A and B. Apparently these acidic peptides, which have a widespread distribution in the endocrine system, are co-stored and therefore also co-secreted with ANP.
Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.
Authors: Silverman A J , Witkin J W , Millar R P
Published: April 2011

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.
Rapid primary microwave-osmium fixation. I. Preservation of structure for electron microscopy in seconds.
Authors: Login G R , Dwyer B K , Dvorak A M
Published: April 2011

Microwave irradiation (MWIr) of tissues immersed in aldehydes has been used to preserve fine structure in seconds. The purpose of this study was to extend these findings to include rapid primary osmium fixation in a microwave (MW) device with a high volume exhaust. Blocks of rat heart and liver were trimmed to approximately 4 mm3 and exposed to 0.2 M symcollidine-buffered 2% osmium tetroxide for a period of 6-7 sec during MWIr (final solution temperature approximately 45 degrees C). We also evaluated rapid fixation of tissues exposed to MWIr simultaneously with immersion in dilute Karnovsky's fixative (6-7 sec to approximately 50 degrees C) followed by MWIr of specimens immersed in osmium (7 sec to approximately 45 degrees C). Tissues were stored in 0.1 M sodium cacodylate buffer (pH 7.3, 4 degrees C) up to 2 weeks and were stained en bloc in uranyl acetate, dehydrated in a graded series of alcohols, and embedded in propylene oxide-Epon sequence. Thin sections were stained with lead citrate and examined by transmission electron microscopy. We demonstrate that fine structural preservation of tissue blocks can be achieved by MWIr in aldehyde and/or osmium in seconds.
Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.
Authors: Sata T , Zuber C , Rinderle S J , Goldstein I J , Roth J
Published: April 2011

The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them.
Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.
Authors: McKeever P E , Wahl R L , Shakui P , Jackson G A , Letica L H , Liebert M , Taren J A , Beierwaltes W H , Hoff J T
Published: April 2011

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.
Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy.
Authors: Michel E , Parsons J A
Published: April 2011

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.
Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids.
Authors: Prasadarao N , Tobet S A , Jungalwala F B
Published: April 2011

Monoclonal antibody (MAb) HNK-1 recognizes a carbohydrate epitope present in certain glycolipids, glycoproteins, and proteoglycans. Five different fixation methods, together with biochemical analyses of the antigens, were evaluated to study immunocytochemical localization of this epitope in layers of adult rat cerebellum; 4% paraformaldehyde/0.5% cetylpyridinium chloride was found to be optimal for overall immunoreactivity, and the antigens were apparent in all cerebellar layers. To differentially localize HNK-1-reactive carbohydrate epitope on proteins vs lipids in cerebellar layers, we tested the effect of 0.2%, 2%, or 4% glutaraldehyde combined with 2% paraformaldehyde (GT/PF) on HNK-1 and other MAb-reactive protein and lipid antigens; 2% or 4% GT/PF significantly reduced or abolished immunoreactivity of MAb HNK-1 and 5F9 (reacting with microtubule-associated protein 2) with cerebellar proteins analyzed on Western blots, but did not decrease HNK-1 reactivity to lipid antigens on HPTLC blots. In cerebellar tissue sections, HNK-1 and 5F9 immunoreactivity was reduced after 2% or 4% GT/PF fixation. However, significant amounts of HNK-1 immunoreactivity remained in molecular layer and deep cerebellar nuclei. GT/PF fixation did not cause significant changes in immunoreactivity patterns of other carbohydrate lipid antigens, such as those that react with MAb A2B5, 7A, and WCC4. Therefore, carbohydrate epitope on lipids, as opposed to that on proteins, may be preferentially detectable by immunocytochemistry after fixation with 2% or 4% GT/PF. The selective localization of HNK-1-reactive carbohydrate in the molecular layer and deep cerebellar nuclei with 2% or 4% GT/PF fixation correlates well with the observed presence of HNK-1-reactive lipids in these areas but not in the granular layer and white matter, as determined by microdissection of the individual layers and biochemical analysis. The application of 2% or 4% GT/PF fixation as a general method for differentiating the same carbohydrate epitope on proteins vs lipids in immunocytochemistry for other tissues and other antibodies remains to be further evaluated.
Ezrin immunoreactivity in neuron subpopulations: cellular distribution in relation to cytoskeletal proteins in sensory neurons.
Authors: Everett A W , Nichol K A
Published: April 2011

Ezrin was first identified as a low-abundance phosphoprotein associated with the cytoskeletal core of microvilli, where it may function as a regulatory protein. We report immunocytochemical evidence for expression of ezrin, or an ezrin-like protein of molecular mass near 80 KD, confined to select populations of neurons, including sensory, motor, and autonomic, during chick embryonic development. We have compared the distribution of anti-ezrin staining with that of other major cytoskeletal proteins in sensory neurons in an effort to identify a possible association of the neural homologue of ezrin with the neuronal cytoskeleton. The diffuse distribution of anti-ezrin staining in the cell soma bore little resemblance to the filamentous staining observed with antibodies to the 68 KD neurofilament protein and alpha-tubulin. F-actin staining with fluorescein-conjugated phalloidin was indistinguishable from the anti-ezrin staining pattern in the soma of cultured neurons, including a peak in staining intensity around the periphery of the cell. Microfilaments in growth cones did not stain with the ezrin antibody. A close correspondence between the anti-ezrin and anti-spectrin staining patterns was found on cryostat sections of dorsal root ganglia, but the anti-spectrin staining was weak and could not be demonstrated in culture. Our findings, primarily from cultured neurons, are not inconsistent with ezrin associating with F-actin, although not with microfilaments found in motile structures such as growth cones.
Polarization of gelsolin and actin binding protein in kidney epithelial cells.
Authors: Hartwig J H , Brown D , Ausiello D A , Stossel T P , Orci L
Published: April 2011

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.
Lysosomal trafficking in rat cardiac myocytes.
Authors: Marjomäki V S , Huovila A P , Surkka M A , Jokinen I , Salminen A
Published: April 2011

By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. MPR was associated mostly with inner membranes, whereas lgp120 was predominantly bound to the outer limiting membrane. MPR, lgp120, and MEP were not detected in Golgi stacks, but some labeling was seen in the putative TGN. Our data suggest that the MPR structures are prelysosomes involved in lysosomal enzyme targeting in rat cardiac myocytes.
Na+, K(+)-ATPase in cat salivary glands and changes induced by nerve stimulation: an immunohistochemical study.
Authors: Winston D C , Schulte B A , Garrett J R , Proctor G B
Published: April 2011

The enzyme Na+, K(+)-ATPase was localized immunohistochemically in major salivary glands of the cat before and after autonomic nerve stimulation. Immunostaining was limited to basolateral plasma membranes. Cells lining striated and excretory ducts contained abundant Na+, K(+)-ATPase and showed no changes with neural stimulation. Serous-type cells in resting glands varied in reactivity, showing weak to moderate staining intensity in the parotid gland and more uniform staining of greater intensity in the sublingual gland. In contrast, demilune cells in the resting submandibular gland showed little if any staining. Mucous-type cells were negative in all glands. Parasympathetic stimulation promoted a gradual increase in immunostaining of submandibular demilune cells, which became marked with time. Sympathetic stimulation produced no detectable changes in Na+, K(+)-ATPase immunoreactivity in any site. These results support the concept that basolateral Na+, K(+)-ATPase is essential to the formation of a near-isotonic primary saliva by serous-type cells. The mechanism whereby parasympathetic stimulation evokes a marked flow of submandibular saliva remains unexplained, but has now been shown to involve a marked increase in the immunoreactivity of Na+, K(+)-ATPase at the base of the gland's demilune cells.
Subcellular localization of blood group substances ABH in human salivary glands.
Authors: Cossu M , Riva A , Lantini M S
Published: April 2011

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.
Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies.
Authors: Otsuki Y , Maxwell L E , Magari S , Kubo H
Published: April 2011

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.
Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues.
Authors: Cordon-Cardo C , O'Brien J P , Boccia J , Casals D , Bertino J R , Melamed M R
Published: April 2011

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.
Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats.
Authors: Vinores S A , McGehee R , Lee A , Gadegbeku C , Campochiaro P A
Published: April 2011

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.
Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography.
Authors: Davidson D A , Bohannon N J , Corp E S , Lattemann D P , Woods S C , Porte D , Dorsa D M , Baskin D G
Published: April 2011

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.
Mechanisms and products of azo coupling in histochemical protease procedures based on primary aromatic amines as unspecific moieties.
Authors: Frank H G
Published: April 2011

It is presumed that the azo dyes generated by histochemical protease reactions are formed by substitution of a reactive aromatic carbon. They are referred to as dyes of the C-azo series. To confirm this assumption, the absorption spectra between 330 and 630 nm of azo dyes resulting from coupling between various aromatic amines of the aniline and naphthylamine series and the diazonium salts Fast Blue B and Fast Garnet GBC were studied in test tube experiments. Some of the amines were blocked by methylation to prevent coupling either at the amino group (N-methylated) or at the aromatic nucleus (C-methylated). Coupling was performed in buffered aqueous solutions of the diazonium salts. For analysis the azo dyes were dissolved in dimethylformamide. For acid rearrangement these solutions were acidified and incubated at elevated temperatures. After detection of dipeptidyl peptidase IV in tissue sections using Gly-Pro-4-methoxy-2-naphthylamine as substrate, the resulting dye was extracted and compared with the test tube compounds. All aromatic amines yielded azo dyes. Dyes extracted from sections and those test tube compounds derived from unmethylated or C-methylated amines showed almost identical spectral maxima, whereas dyes formed by N-methylated amines yielded different spectra. Acid rearrangement did not influence the spectral maxima of the N-methylated amine-derived dyes. Dyes resulting from C-methylated amines were destroyed. The results indicate that under histochemical conditions diazonium salts react primarily with the liberated free amino group but not with the aromatic nucleus of the unspecific moiety. Therefore, it is proposed that the formula of the final reaction product in naphthylamine-based protease histochemistry should be given as an N-azo dye, e.g., as a triazene.
Localization of cathepsin B in two human lung cancer cell lines.
Authors: Erdel M , Trefz G , Spiess E , Habermaas S , Spring H , Lah T , Ebert W
Published: April 2011

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.
Computer analysis of double labeling: ultrastructural distribution of vasoactive intestinal peptide and transferrin in human colon cancer cells.
Authors: Lefevre C , Phan H H , Anteunis A , Rosselin G
Published: April 2011

Transferrin (Tf) and vasoactive intestinal peptide (VIP) were labeled with horseradish peroxidase (HRP) and 125I, respectively. To determine whether two simultaneously incubated ligands are conveyed by the same population of endosomal vesicles in human colon carcinoma cells (HT-29), we used an analysis system derived from the cross-fire method for quantitation of autoradiographic data. This system permitted the collection of data and the statistical calculations required by the double labeling of the cells. HRP-labeled Tf organelles were chosen as reference structures of the endosomal apparatus and taken as the conventional source of the radiolabeling. Our data established from the co-localization hypothesis strongly suggest that after a 30-sec (T 1/2) and a 10-min (T10) internalization at 37 degrees C, VIP and Tf share in major part the same endocytic pathway and even the recycling route to the cell surface. At T10, most of the radiolabeling was located inside the tubulovesicular network, and we also detected slight radiolabeling inside the vesicles recycling Tf. The number of double-labeled endosomes involved in ligand traffic were advantageously observed with our computer-assisted analysis system.
Peptidylglycine alpha-amidating monooxygenase (PAM) in Schwann cells and glia as well as neurons.
Authors: Rhodes C H , Xu R Y , Angeletti R H
Published: April 2011

We raised an antiserum against the synthetic peptide FKETTRSFSNECLGTTR corresponding to the amino terminus of the enzyme peptidylglycine alpha-amidating monooxygenase (PAM). Control experiments were performed to determine the specificity of the antiserum and its suitability for the immunohistochemical identification of PAM-containing cells. An immunoaffinity column made with the antibody coupled to Sepharose permitted the isolation of the active enzyme. Peptide-agarose immunoadsorbant removed the antibodies responsible for the characteristic staining patterns in immunohistochemical experiments. As expected from the widespread distribution of amidated peptides in the nervous system, PAM immunoreactivity was detected in perikarya in a variety of locations, including the pituitary, the hypothalamic periventricular and supraoptic nuclei, neocortex, and sensory ganglia. Punctate immunostained fibers, especially around neuronal perikarya, were observed in regions known to receive amidated peptidergic afferents. In addition, PAM immunoreactivity was observed in some neurons not known to produce amidated peptides (e.g., pyramidal cells of the hippocampus). This result suggests that these neurons also produce an amidated peptide. PAM immunoreactivity was also detected in several unexpected cell types, including ependyma, choroid plexus, oligodendroglia, and Schwann cells. The presence of enzymatically active PAM in Schwann cells was confirmed by measurements of amidating activity in ligated and control sciatic nerve. These results suggest that these non-neuronal cells may produce amidated peptides.
Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary.
Authors: Bassetti M , Huttner W B , Zanini A , Rosa P
Published: April 2011

We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.
Immunoelectron microscopic study of a new D-amino acid oxidase-immunoreactive subcompartment in rat liver peroxisomes.
Authors: Usuda N , Yokota S , Ichikawa R , Hashimoto T , Nagata T
Published: April 2011

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, 3-ketoacyl-CoA thiolase, L-alpha-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), was assayed. The electron-lucent area was negative for all of these. By double staining for D-amino acid oxidase and catalase, using colloidal gold particles of different sizes, these enzymes were shown to be located in separate areas in the matrix.
Morphometric analysis of gold particles in low-label cell compartments.
Authors: Hammel I , Kalina M
Published: April 2011

Quantitative estimation of the binding of gold-conjugated ligands to various cell organelles has become a commonly used method to quantify the amount of ligand-binding sites associated with those organelles. However, often a small percentage of organelles is labeled or the density of gold labeling is low. We have defined the "gold-labeled region" as a zone that has a boundary defined by the localization of the outermost gold particles. Such a phenomenon was recently observed in a study of the internalization of gold-labeled native surfactant into lamellar bodies of cultured pulmonary type II cells. We estimated the size and density of gold-labeled regions in lamelar bodies using a simple stereological approach and demonstrated that the low percentage of gold-labeled organelles can be explained as a result of the probability of random selecting through the labeled areas. Our method, which permits use of transmission electron microscopy to calculate the true parameters of gold-labeled regions, can significantly facilitate analyses of ligand binding to various cell compartments.
A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.
Authors: Boerjan M L , Baarends W M , Ruven H J
Published: April 2011

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.
A new technique to identify hybrid myotubes in vitro without culture fixation.
Authors: Labrecque C , Huard J , Dansereau G , Robitaille L , Tremblay J P
Published: April 2011

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.
Comparison of 35S and biotin as labels for in situ hybridization: use of an HPV model system.
Authors: Unger E R , Hammer M L , Chenggis M L
Published: April 2011

Colorimetric in situ hybridization is a method of potential importance in diagnosis and research. The largest criticism of the method has been a perceived loss of sensitivity compared with autoradiographic techniques. Our more positive experience with automation of colorimetric in situ hybridization led us to undertake a direct comparison of the sensitivity of 35S- and biotin-labeled probes. Serial sections of formalin-fixed, paraffin-embedded cell pellets from four human cervical carcinoma cell lines with known copies of HPV (CaSki, 400-600 copies HPV 16; HeLa, 10-50 copies HPV 18; SiHa, 1-2 copies HPV 16; HTB31, no known copies HPV) were hybridized with protocols optimized for autoradiographic or colorimetric detection. Both methods gave comparable results, with differences in each technique seen at the limits of sensitivity. The 1-2 copies of HPV 16 per SiHa cell can be detected with both methods; however, grain counting is required for interpretation of the autoradiographic result. This degree of sensitivity for colorimetric in situ hybridization in formalin-fixed, paraffin-embedded material is achieved through careful optimization of probe size and labeling, adequate tissue digestion, and removal of background. Autoradiography may be preferred in situations where quantitation is required, but colorimetric detection retains the advantages of speed, potential for automation, and improved localization of signal with comparable sensitivity.