bluuebunny/crossref_metadata_2025 huggingface dataset:
AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle.
Authors: Thompson J L , Sabina R L , Ogasawara N , Riley D A
Published: April 2011

The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.
Poor reliability of immunocytochemical localization of IgG in immersion-fixed tissue from the central nervous system.
Authors: Fabian R H
Published: April 2011

The effect of fixation technique and post mortem-to-fixation interval in immersion-fixed tissue from the central nervous system on immunocytochemical staining for the presence of an immunoglobulin was determined in mice. Immersion-fixed tissue was found to be inferior to perfusion-fixed tissue for immunocytochemical staining of this serum protein. Unlike what has been observed for other antigens, the quality of staining for IgG in immersion-fixed tissue decreased to unacceptable levels if the post mortem-to-fixation interval was increased to more than a few hours. This effect may be secondary to the rapid post-mortem disintegration of the blood-brain barrier and a resulting diffusion of serum proteins into surrounding tissue from the vasculature.
Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.
Authors: Carter J H , Richmond R E , Carter H W , Potter C L , Daniel F B , DeAngelo A B
Published: April 2011

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.
Simultaneous immunostaining method for localization of bromodeoxyuridine and calcitonin gene-related peptide.
Authors: Montuenga L M , Springall D R , Gaer J , McBride J T , Polak J M
Published: April 2011

A new simultaneous double immunostaining method has been optimized to localize the DNA synthesis marker bromodeoxyuridine (BrdU) and calcitonin gene-related peptide (CGRP) in endocrine cells of Bouin's-fixed, paraffin-embedded rat lung. Nuclease pre-treatment before immunostaining is compatible with optimal tissue morphology and CGRP antigenicity preservation. Nickel-enhanced development of avidin-biotin-peroxidase staining is used to show CGRP immunoreactivity in black and alkaline phosphatase-anti-alkaline phosphatase is applied to demonstrate incorporated BrdU in red. The present methodology could be useful for studies requiring detection of incorporated BrdU in cells producing regulatory peptides or other labile antigens.
Distribution of the extracellular matrix proteins tenascin, fibronectin, and vitronectin in fetal, infant, and adult human spleens.
Authors: Liakka K A , Autio-Harmainen H I
Published: April 2011

Using immunohistochemistry, we investigated the distribution of the extracellular matrix (ECM) glycoproteins tenascin, fibronectin, and vitronectin in fetal [16-24 gestational weeks (GW)], infant (40 GW), and adult human spleens to clarify the presence of these proteins during different phases of maturation. In the red and white pulp, tenascin and fibronectin were constant components of the reticular fibers from the age of 18 GW onwards, whereas vitronectin was seen only in adult spleens. The immunohistochemical staining patterns of tenascin and fibronectin remained unchanged at different fetal ages. Ring fibers, which are modified basement membranes around venous sinuses, became visible relatively late, and in adult spleens they contained both tenascin and vitronectin but lacked fibronectin. The composition of the ring fibers is therefore clearly different from that of ordinary basement membranes, which have not been reported to contain tenascin or vitronectin. The rapidly increasing number of reticular fibers in the spleen at the age of approximately 18 GW corresponds with the beginning of lymphatic colonization. Reticular fibers, rich in ECM glycoproteins, form a framework to which cells can migrate and attach. We suggest that the composition of these fibers might be important for lymphatic colonization and function of the spleen.
Atrial natriuretic peptide-like immunoreactivity in astrocytes of parenchyma and glia limitans of the canine brain.
Authors: McKenzie J C
Published: April 2011

The Na(+)-regulating and vasoactive peptide hormone atrial natriuretic peptide (ANP) has been localized in cultured glial cells from neonatal rat brain but has not been reported in situ in adult brain. In the present study, immunohistochemical localization of ANP in astrocytes of adult canine brain was obtained using an antibody against rat ANF IV, a 25-amino acid fragment identical to low molecular weight brain ANP. ANP-like immunoreactivity was localized in widely dispersed astroglia throughout the canine brain, including Types I and II, in addition to some astrocytes of the glia limitans (GL).
Demonstration of dopamine-immunoreactive cells in the gastrointestinal tract of gerbils (Meriones unguiculatus).
Authors: Dawirs R R , Teuchert-Noodt G , Kampen W U
Published: April 2011

We investigated dopamine immunoreactivity in the small intestine of gerbils (Meriones unguiculatus), using a sensitive and selective antibody against glutaraldehyde-conjugated dopamine. Dopamine-immunoreactive cells were found within the mucosal epithelium along the entire extent of the small intestine. Labeled cells were regularly distributed in the upper half of the intestinal villi, basally attached to the basement membrane and always reaching as far as the epithelial surface. Dopamine-containing cells revealed a spindle-like shape, and both light and electron microscopic characteristics relate them to typical open-type gut endocrine cells. Thus, this current study directly identified dopamine as a probable secretory product in basal granulated cells of the small intestine. The functional significance of these cells is discussed in relation to the current view of dopaminergic actions in peripheral tissues.
Detection of proliferating cell nuclear antigen in diagnostic histopathology.
Authors: Wolf H K , Dittrich K L
Published: April 2011

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.
Immunocytochemical localization of histamine in secretory granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique.
Authors: Login G R , Galli S J , Dvorak A M
Published: April 2011

We used a post-embedding immunogold labeling approach to define the fine-structural localization of histamine in rat peritoneal mast cells that were fixed using either standard aldehyde fixation or a fast microwave-aldehyde fixation method. Specimens were processed routinely for electron microscopy. Thin sections were exposed first to guinea pig antihistamine antiserum and then to gold-conjugated goat IgG directed against guinea pig IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with non-immune sera did not show labeling of mast cells. Adsorption of antihistamine antiserum with purified histamine or histamine bound to agarose showed a significant reduction (p less than 0.005) in granule staining. We also confirmed that our isolation procedures yielded functionally competent mast cells which released histamine when stimulated with sheep anti-rat IgE antiserum or with compound 48/80. These studies define the conditions of fixation for electron microscopy that are appropriate for the localization of histamine in the granule matrix of rat peritoneal mast cells.
Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.
Authors: Hermiston M L , Latham C B , Gordon J I , Roth K A
Published: April 2011

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.
An infrared study of the interaction of polymethyl methacrylate with the protein and mineral components of bone.
Authors: Pleshko N L , Boskey A L , Mendelsohn R
Published: April 2011

We used Fourier transform infrared (FT-IR) microscopic mapping techniques to investigate the infiltration of polymethyl methacrylate (PMMA), a widely used medium for embedding biological tissues, into rat femur sections. Monitoring of the infrared absorbances of the PMMA carbonyl stretch, the protein amide I, and the apatite mineral phosphate stretch over a 225 x 975-microns region of the epiphyseal growth plate region of the rat femur enabled comparison of the relative amount of each component in distinct regions of the tissue. It was found that PMMA penetrates less into regions of greater mineral density and that the frequency of the PMMA carbonyl absorbance from the embedded tissue, 1729 cm-1, is identical to the free PMMA carbonyl frequency. This is consistent with a diffusion mechanism of infiltration of the PMMA, with no specific chemical interaction between the PMMA and the tissue components.
Differential visualization of cholinesterasic neuronal somata and fibers by use of modifications of acetylcholinesterase pharmacohistochemistry.
Authors: Di Patre P L , Mathes C W , Butcher L L
Published: April 2011

We modified existing acetylcholinesterase (AChE) histochemical techniques to visualize cholinergic neuronal somata and processes in the same rat brain. The AChE staining procedure of Karnovsky and Roots, combined with diisopropylfluorophosphate (DFP) pre-treatment, have previously allowed visualization of neuronal somata. Fibers have been visualized by nickel-diaminobenzidine (DAB)-hydrogen peroxide histochemical intensification. We combined novel modifications of these techniques to visualize cell bodies and fibers simultaneously. Maximal staining of neuronal somata occurs with a 1:1 dilution of both the Karnovsky-Roots medium and the nickel-DAB solution; diluting the Karnovsky-Roots medium 1:10 and increasing the concentration of the nickel-DAB solution twofold allows visualization of fibers previously unstainable with DFP pre-treatment and the classical AChE protocol. It is now possible to visualize both neuronal somata and fibers in adjacent sections of the same brain, in a quick and inexpensive manner.
Arachidonate 12-lipoxygenase is localized in neurons, glial cells, and endothelial cells of the canine brain.
Authors: Nishiyama M , Watanabe T , Ueda N , Tsukamoto H , Watanabe K
Published: April 2011

12-Lipoxygenase oxygenates the 12 position of arachidonic acid, producing its 12S-hydroperoxy derivative. We have been interested in the distribution of 12-lipoxygenase in the central nervous system. Previously, by the use of a monoclonal anti-12-lipoxygenase antibody, we proved that one of the arachidonate 12-lipoxygenases existed in canine brain. The present study was therefore designed to elucidate the exact immunohistochemical localization of arachidonate 12-lipoxygenase in canine brain, using a polyclonal anti-12-lipoxygenase antibody. Canine brains were irrigated thoroughly with ice-cold heparinized saline and phosphate-buffered saline to remove blood cells, and then were fixed in 4% periodate-lysine-paraformaldehyde at 4 degrees C for 3 hr. Immunostaining by the indirect method with the peroxidase-labeled antibody was performed for light microscopic observation. The immunohistochemical study demonstrated that many neurons and glial cells in the cerebrum, basal ganglia, and hippocampus were positively stained. Biochemical results as described previously and those of immunohistochemistry indicate that 12-lipoxygenase is definitely localized in various brain parenchymal cells.
Application of an image analyzer to gold labeling in immunoelectron microscopy to achieve better demonstration and quantitative analysis.
Authors: Shimizu H , Nishikawa T
Published: April 2011

An image analyzer was applied to pre- and post-embedding immunogold labeling with 5-nm gold probes in electron micrographs of several skin basement membrane antigens to improve the visualization of immunolabeling. With a TV camera connected to a color image analyzer, an image of an original immunoelectron micrograph was projected on a TV screen. The image was recorded in the analyzer as Record 1. After the floating threshold method procedure to reduce the contrast of the skin structure, electron-dense 5-nm gold particles could be easily detected. With the analyzer, these particles were then suitably enhanced in color and in size and their image was recorded as Record 2. Records 1 and 2 were then overlapped on the TV screen to build up a double-image picture. Compared with the small, electron-dense 5-nm gold particles in the original electron micrograph, ultrastructural localization of bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and the collagenous part of Type VII collagen were more clearly and easily seen, even on low-magnification electron micrographs. The level of background labeling could also be accurately and objectively evaluated. By counting the number of gold particles labeling a certain area and using the analyzer to interpret the result as a diagram, quantitative analysis was also possible. We believe that this technique should be widely applicable to immunogold electron microscopy, not only of skin but also of other substrates of interest.
Divergent and co-localization of the two small proteoglycans decorin and proteoglycan-100 in human skeletal tissues and tumors.
Authors: Bosse A , Schwarz K , Vollmer E , Kresse H
Published: April 2011

The core protein of a recently described small proteoglycan, proteoglycan-100, was localized in fetal human tissues by indirect immunocytochemistry and compared with the localization of known members of the small proteoglycan family. Co-localization of decorin and proteoglycan-100 was seen in bone tissue but decorin and proteoglycan-100 exhibited a substantially divergent distribution in fetal skin, cartilage and in the mineralization zone of the growth plate. Proteoglycan-100 was also found in striated muscle, nerve fibers, and synovial tissue. Immunostaining of a chondroblastic osteosarcoma demonstrated chondroid cells selectively expressing either proteoglycan-100 or decorin. Co-expression of both small proteoglycans was observed in sections from a chordoma. In fetal bone and in the two tumors, colocalization of proteoglycan-100 and of biglycan was also found. These results provide evidence of the wide and characteristic distribution of proteoglycan-100.
Novel monoclonal antibodies specific for human cardiac myosin light-chain 1: useful tools for analysis of normal and pathological hearts.
Authors: Fujimoto K , Yasue H , Nakao K , Yamamoto H , Hitoshi Y , Jougasaki M , Okumura K , Ogawa H , Takatsu K , Miyamoto E
Published: April 2011

To investigate the developmental, physiological and pathophysiological roles of human cardiac myosin light-chain 1 (LC1s), we developed two novel monoclonal antibodies (KA1 and KB1) against human cardiac LC1s and examined LC1s in normal and pathological hearts immunohistochemically. KA1 and KB1 were specific only for atrial LC1 (ALC1) and for both ALC1 and ventricular LC1 (VLC1), respectively, in human hearts. Among human tissues tested, including skeletal muscle, vascular smooth muscle, and liver, KA1 did not crossreact with proteins in any other tissues than atria, whereas KB1 crossreacted with the slow-type LC1 of skeletal muscle. Among adult mammalian hearts of several other species including pig, dog, hamster, and rat, KA1 and KB1 crossreacted only with ALC1 and with both ALC1 and VLC1, respectively. ALC1 was strongly and uniformly observed in human fetal atria and ventricles and in normal adult human atria, but sporadically in normal adult human ventricles. In the overloaded ventricle (dilated cardiomyopathy), ALC1 was highly augmented but not uniform. These results suggest that the fetal VLC1 is immunohistochemically identical to the adult type of ALC1 and that ALC1 is expressed homogeneously in human fetal ventricles and sporadically in normal adult ventricles, and is re-expressed heterogeneously and in an increased amount in the overloaded ventricle.
Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro.
Authors: Kalina M , McCormack F X , Crowley H , Voelker D R , Mason R J
Published: April 2011

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar Type II cells. In the present study we followed the internalization and intracellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar Type II cells. Internalization of native rat SP-A was compared with that of recombinant rat and human SP-A isolated from a patient with alveolar proteinosis. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active, as demonstrated by inhibition of phospholipid secretion from alveolar Type II cells. The SP-A-gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron-lucent and -dense multivesicular bodies. Labeling of LB was time dependent, and after 7 hr 30-40% of these organelles were labeled. Alkylation of SP-A greatly reduced internalization, as did an excess of non-conjugated SP-A. No qualitative differences in uptake were observed with the three forms of SP-A. The percent of labeled LB was similar (30-40%) after 7 hr of internalization with the three species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaccharide, but these features did not affect its internalization or the rate of LB labeling. Internalization of the gold-conjugated SP-A and endocytosis of the fluid-phase marker Lucifer Yellow were related to the shape of Type II cells. Both uptake of SP-A, which is receptor mediated, and fluid-phase endocytosis were found to be less active in the flattened than in the rounded cells. Therefore, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling. However, it is possible that both morphology and decreased endocytosis are independent manifestations related to the loss of differentiated function of cultured Type II cells.
Keratan sulfate and dermatan sulfate proteoglycans associate with type VI collagen in fetal rabbit cornea.
Authors: Takahashi T , Cho H I , Kublin C L , Cintron C
Published: April 2011

Keratan sulfate proteoglycan (KSPG) and dermatan sulfate proteoglycan (DSPG) are associated with collagen fibrils in adult rabbit cornea. Because certain cytochemical data suggested that proteoglycans are associated with type VI collagen in the fetal rabbit cornea, we developed polyclonal antibodies specific to the core proteins of rabbit corneal KSPG (lumican and/or fibromodulin) and DSPG (decorin and/or biglycan) and used the antibodies as immunocytochemical probes to determine proteoglycan ultrastructural location. Immunogold particles were associated with Type VI collagen filaments but not with collagen fibrils in fetal and neonate rabbit cornea. Association of corneal KSPG and DSPG with Type VI collagen was immunocytochemically confirmed with monoclonal antibodies to low-sulfated keratan sulfate glycosaminoglycan (GAG) and chondroitin-4-sulfate GAG of DSPG. The monospecificity of the polyclonal and monoclonal antibodies and exclusive binding of antibodies to Type VI collagen filaments, together with previous chemical identification of low-sulfated KSPG and DSPG in developing rabbit cornea, support our contention that corneal proteoglycans in developing rabbit cornea are associated with Type VI collagen.
Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization.
Authors: Reed M J , Puolakkainen P , Lane T F , Dickerson D , Bornstein P , Sage E H
Published: April 2011

SPARC and thrombospondin 1 (TSP-1) are secreted glycoproteins expressed by similar types of cells in culture and in tissues. To compare these two proteins in vivo, we analyzed the differential expression of SPARC and TSP-1 during wound repair. Full-thickness incision wounds were made in rats and biopsied at 12 hr-14 days. Antibodies against SPARC revealed an increased proportion of immunoreactive fibroblastic cells at the wound edge at 3 days with maximal numbers at 7 days. In situ hybridization for SPARC produced results consistent with those of immunohistochemistry. With combined immunohistochemistry and in situ hybridization, some of the macrophages at the wound edge expressed SPARC mRNA. In contrast, immunoreactivity for TSP-1 was extracellular; expression at the wound edge was noted at 12 hr and was maximal at 1-2 days. TSP-1 mRNA was found in the thrombus, but not at the wound edge. In conclusion, SPARC and TSP-1 have contrasting roles during wound healing. SPARC expression from the middle through late stages of repair was consistent with its previously proposed functions in remodeling; in contrast, the transient expression of TSP-1 early in repair might facilitate the action of other proteins in recruitment and/or proliferation of cells in the healing wound.
Endothelin in normal lung tissue of newborn mammals: immunocytochemical distribution and co-localization with serotonin and calcitonin gene-related peptide.
Authors: Seldeslagh K A , Lauweryns J M
Published: April 2011

We demonstrated immunoreactivity for endothelins (ET)-1, -2, and -3 and for the precursor, big-ET-1, in the pulmonary diffuse neuroendocrine system (PDNES) of newborn cat, rat, hamster, and mouse. ET-like positive neuroepithelial bodies (NEB) were numerous in the intrapulmonary airways and the alveolar parenchyma. Single neuroendocrine cells (NEC) were less often labeled and mainly localized in the larger bronchi. ET-3-reactive neuronal elements were rarely observed. The intensity and number of immunostained NEB were highest for ET-3, followed in declining order by big-ET-1, ET-1, and ET-2. ET-like possessing NEB displayed interspecies differences. We conclude that ET-3 represents a neuroendocrine form of the ET peptide family. NEB expressing several ET isoforms can be grouped into NEB containing either big-ET-1 and ET-1 or ET-3 only. ET-like immunoreactivity was present in a subpopulation of serotonin (5HT)- and/or calcitonin gene-related peptide (CGRP)-positive NEB. As ET, 5HT, and CGRP have potent pulmonary vaso- and/or bronchomotor effects, our observations suggest that they play a separate or synergistic role in regulatory function of the mammalian PDNES, exerting their influence by paracrine, endocrine, and neurocrine pathways or a combination of these.
Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections.
Authors: Lin N S , Chen C C , Hsu Y H
Published: April 2011

We developed an in situ hybridization technique for localization of plant virus RNA in ultra-thin sections of virus-infected, Lowicryl HM20- or osmium-fixed, Araldite-embedded plant leaf tissues. A digoxigenin-labeled in vitro transcript corresponding to 173 nucleotides at the 3' end of bamboo mosaic virus (BaMV) RNA was used as a riboprobe and the hybrids were detected by incubation with sheep antidigoxigenin antibody followed by gold-labeled rabbit anti-sheep IgG. Pre-treatment of ultra-thin sections with an etching agent was the critical step in enhancing hybridization signals on Lowicryl-embedded thin sections. Etching combined with K-metaperiodate treatment increased the accessibility of nucleic acids to riboprobes in osmicated Araldite-embedded sections. BaMV RNA was specifically detected within chloroplasts, mitochondria, and nuclei of infected cells. BaMV virions and BaMV-specific electron-dense crystalline bodies were also labeled. The labeling intensity on Lowicryl-embedded samples, in general, was much higher than that on osmicated Araldite-embedded samples. However, our procedure offers the advantage that it permits labeling of viral nucleic acids in tissues already processed for routine EM and should be applicable for in situ labeling of any cellular nucleic acids.
Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues.
Authors: Breuss J M , Gillett N , Lu L , Sheppard D , Pytela R
Published: April 2011

Integrins are cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Each integrin consists of two glycoprotein subunits (alpha and beta). We have previously described a novel integrin beta-subunit, beta 6, which is expressed in cultured epithelial cells. beta 6 can associate with alpha v to form the fibronectin-binding heterodimer alpha v beta 6. Here we report the tissue distribution of beta 6 integrin mRNA determined by in situ hybridization of a beta 6 cRNA probe with representative frozen tissue sections from a rhesus monkey tissue bank. We detected beta 6 mRNA exclusively in epithelial cells. However, beta 6 mRNA expression varied greatly among different epithelia. High levels of beta 6 mRNA were found only in two very specialized epithelial cell types: a portion of the kidney tubule epithelium, termed macula densa, and the endometrial epithelium of secretory phase uterus. In the endometrium, beta 6 expression was highest in the differentiated epithelium of functional layer glands, suggesting that beta 6 expression can be regulated in a differentiation-dependent manner. beta 6 expression may also depend on the stage in the estrous cycle, since we found much lower beta 6 mRNA levels in a specimen of proliferative phase endometrium. Epithelium in several other tissues, including salivary gland ducts, gall bladder, and epididymis, contained detectable levels of beta 6 mRNA, albeit much lower than in macula densa and endometrium. In other epithelia, including skin and lung, beta 6 mRNA was undetectable. Taken together, these results suggest that in normal adult primates beta 6 expression is regulated in a cell type-specific manner, restricted to a few epithelial tissues.
Histochemical localization of copper in the intestine and kidney of macular mice: light and electron microscopic study.
Authors: Kodama H , Abe T , Takama M , Takahashi I , Kodama M , Nishimura M
Published: April 2011

Menkes' disease is an X-linked recessive disorder characterized by accumulation of copper in various organs and cells, such as the intestine, kidney, and cultured fibroblasts. Light and electron microscopic localization of Cu was investigated in the intestine and kidney of macular mice, an animal model of Menkes' disease, by a modified sulfide-silver method. Cu was accumulated in the cytoplasm of the absorptive epithelial cells, the vascular endothelium, and secretory granules of the Paneth cells. In kidney the distal tubule cells and glomeruli of both macular and control mice stained faintly, whereas the organelle-free cytoplasm in the proximal tubule cells of macular mice stained more intensely than those of controls. The nuclei, mitochondria, and lysosomes of the cells of macular mice hardly stained at all. These findings indicate that Cu is concentrated in the organelle-free cytoplasm of the affected cells of macular mice. This suggests that the Menkes' mutation affects Cu transport from the cytosol to the organelles in the cell.
Use of fluorescent latex microspheres (FLMs) to follow the fate of transplanted myoblasts.
Authors: Satoh A , Huard J , Labrecque C , Tremblay J P
Published: April 2011

A potential treatment for Duchenne muscular dystrophy (DMD) is injection of normal myoblasts into dystrophic muscles to induce formation of muscle fibers. To develop this therapy it is important to identify the injected myoblasts and the muscle fibers that they form in the host muscles. Fluorescent latex microspheres (FLMs) were used for this purpose in this study. Normal myoblasts were labeled with FLMs and injected into dystrophin-deficient (mdx) mice. The FLMs clearly indicated the location of injected myoblasts in the host muscle. Muscle fibers containing dystrophin were localized by immunofluorescence and immunoperoxidase. They were observed in clusters near the myoblasts labeled with FLMs. FLMs were also observed in some of these dystrophin-positive fibers in each cryostat section. These results indicate that: labeling myoblasts with FLMs can be used to trace the injected myoblasts in the muscle and to identify the muscle fibers that they formed; injected myoblasts remain near the injected site and do not migrate very far; most of the dystrophin-positive muscle fibers around the injected myoblasts result from fusion of the injected myoblasts; and the low percentage of dystrophin-positive muscle fibers is likely related to limited diffusion and lack of fusion of many injected myoblasts.
Specific immunostaining for CCP II, a novel calcitonin carboxyl terminal peptide encoded by the calcitonin/CGRP gene.
Authors: Giscard-Dartevelle S , Ghillani P , Taboulet J , Troalen F , Segond N , Lasmoles F
Published: April 2011

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way.
Differential expression of lectin-binding sites defines mouse intestinal M-cells.
Authors: Clark M A , Jepson M A , Simmons N L , Booth T A , Hirst B H
Published: April 2011

We investigated the binding of four lectins to the follicle-associated epithelium (FAE) overlying fixed mouse small intestinal Peyer's patches to identify M-cell-specific surface markers. Wheat germ agglutinin and peanut agglutinin displayed heterogeneous staining patterns, binding most avidly to the intestine goblet cells. In contrast, the lectins Ulex europaeus 1 (UEA 1) and Psophocarpus tetragonolobus (winged bean; WBA) were almost exclusively M-cell specific. When confocal laser scanning images of tissues stained with fluorescein isothiocyanate (FITC)-conjugated UEA1 or WBA were compared with the appearance of the same tissues under the scanning electron microscope (SEM), UEA1 strongly stained 97.2% (106/109) of M-cells, 0.6% (3/516) enterocytes, and 0% (0/28) goblet cells, whereas WBA stained 100% (83/83) M-cells, 1.7% (6/361) enterocytes, and 5.3% (1/19) goblet cells. The M-cell specificity of the lectin binding was further demonstrated by localization of horseradish peroxidase (HRP)-conjugated lectins under the transmission electron microscope (TEM). This is the first demonstration of carbohydrates in the glycocalyx of M-cells that are not expressed elsewhere on the FAE surface. These carbohydrates not only provide a means to identify mouse M-cells by LM but may also contribute to the occurrence of specific interactions between microorganisms and the M-cell apical membrane.
The DAB-Mn++ cytochemical method revisited: validation of specificity for superoxide.
Authors: Steinbeck M J , Khan A U , Appel W H , Karnovsky M J
Published: April 2011

We wished to assess whether the previously developed 3,3'-diaminobenzidine (DAB)-Mn++ cytochemical method, purportedly specific for superoxide localization, is detecting superoxide O2.- and/or the superoxide product, O2(1 delta g). We show here that polymorphonuclear leukocytes (PMNs) produce O2(1 delta g) extracellularly in response to non-phagocytic stimuli and that this production is inhibited by addition of superoxide dismutase, an enzyme typically used to demonstrate that a reaction is mediated by O2.-. Because O2(1 delta g) is highly reactive and can be generated from O2.-, the reactivity of a pure chemical source of O2(1 delta g) with the cytochemical probe DAB was examined in the presence and absence of Mn++. Reactions between DAB and O2(1 delta g), thermally released from 1,4-dimethyl-napthalene-1,4-endoperoxide (DNE), indicated that O2(1 delta g) directly reacted with DAB, forming an insoluble DAB polymer, and that this reaction was increased by the presence of Mn++. The direct reaction of O2(1 delta g) with DAB was confirmed using near-IR emission spectroscopy. The near-IR emission spectrum of DNE as it was warmed showed the characteristic energy emission peak of O2(1 delta g) and the intensity of this peak was reduced by the addition of DAB; kq = 1.7 x 10(8) M-1 sec-1. The requirement of Mn++ for oxidation of DAB by O2.- was reconfirmed using potassium superoxide as a pure chemical source of O2.-. In cell studies, however, DAB deposits were not observed in PMNs stimulated under conditions that lead to O2(1 delta g) production [e.g., 0.040 or 0.162 microM 4B-phorbol-12-myristate-13-acetate (PMA)], regardless of whether Mn++ was present in the cytochemical medium. Nor were DAB deposits found in cells stimulated with PMA in the absence of Mn++ or in unstimulated PMNs. Only cells incubated in cytochemical medium containing Mn++ and stimulated to produce large amounts of O2.- (e.g., 3.24 microM PMA) contained DAB deposits. In summary, the DAB-Mn++ cytochemical method remains an excellent method for localizing the production sites of O2.-, since the concentration of O2(1 delta g) within vesicles of stimulated cells is too low to directly oxidize DAB to an electron-dense deposit.
Ultrastructural and antigenic preservation of a delicate structure by cryopreparation: identification and immunogold localization during biogenesis of a secretory component (membrane-matrix connection) in Paramecium trichocysts.
Authors: Momayezi M , Habermann A W , Sokolova J J , Kissmehl R , Plattner H
Published: April 2011

Ultrastructure and antigenicity of the "mesh-like sheath" (MLS), a very delicate structure connecting the membrane and the paracrystalline matrix of Paramecium trichocysts, are well preserved after cryofixation (rapid freezing followed by freeze-substitution in methanol and embedding in Lowicryl K11M at 213K). The MLS is labeled by colloidal gold-bound antibodies (Ab-Au10nm) with primary antibody (Ab) against trichocyst components obtained by recloning hybridoma cells twice. We prepared Western blots from reduced gels obtained from subfractionated trichocysts. Trichocyst membranes displayed reactive bands of 68-70, 63-66, 43, 40 (strongest), and 57 and 54 KD, with a weak band of 38 KD. One of the most abundant protein bands of soluble secretory components (56-57 KD) was also strongly stained on blots. On ultra-thin sections pre-trichocysts display Ab-reactive material concentrated below the trichocyst membrane before the MLS can be recognized as a structural entity. Quantitative evaluation of Ab-Au10-labeled ultrathin sections also revealed passage of MLS materials through the very inconspicuous Golgi apparatus. This was substantiated by Ab-peroxidase labeling. We conclude that MLS components (whose ultrastructure is difficult to preserve) are largely membrane-associated, partly soluble proteins. They form a connection (released during exocytosis) between the abundant paracrystalline matrix components and the organelle membrane. MLS might thus maintain a peripheral aqueous space of functional importance.
Monoclonal antibodies to type VI collagen demonstrate new tissue augmentation of a collagen-based biomaterial implant.
Authors: Werkmeister J A , Tebb T A , White J F , Ramshaw J A
Published: April 2011

We developed a panel of highly specific monoclonal antibodies (MAb) to either human or dog collagen Type VI. Various degrees of species crossreactivities were found with ELISA and immunohistology. Because of these differentiating species specificities, which allowed distinction between the original donor collagen and newly formed host collagen, the MAb proved to be valuable tools in examination of explanted samples of an ovine composite vascular prosthesis retrieved from a canine model. With an MAb that reacts with dog but not sheep collagen Type VI, newly synthesized pockets of collagen Type VI could readily be detected within the prosthesis as early as 3 months after implantation. These areas were associated with regions of cell infiltration, presumably derived from the host. This association was also apparent in the newly formed intimal region of the prosthesis where only host cells were found. Another of the MAb, which was positive against human but not sheep collagen, was also used to demonstrate marked deposition of host collagen Type VI in a retrieved human sample of the prosthesis. In this case the antibody was able to detect collagen on a formalin-fixed tissue, which would broaden the scope of its use in clinical and pathological situations. Use of these novel antibody probes provides a rapid marker for new tissue augmentation of implanted biological devices which would be an indicator of the long-term performance of a prosthesis.
Quantitative electron probe microanalysis of acetylcholinesterase activity in rat brain sections.
Authors: Pogorelov A G , Budantsev AYu  , Pogorelova V N
Published: April 2011

In this study we determined acetylcholinesterase (AChE) activity by electron probe microanalysis (EPMA). AChE was localized in rat brain sections by a histochemical reaction that produced a Cu-ferricyanide precipitate. This reaction is based on the hydrolysis of acetylcholine substrate with AChE. The copper (iron) concentration in the deposit is proportional to acetylcholine involved in the hydrolysis reaction. Absolute determination of the size of the reaction was calculated from EPMA measurements compared with those made from a dextran standard containing a known amount of Cu(Fe). Electron probe microanalysis allows AChE activity to be assayed quantitatively on thick brain sections with a spatial resolution of 10 microns.
Cellular localization of endothelial alkaline phosphatase reaction product and enzyme protein in the myocardium.
Authors: Schultz-Hector S , Balz K , Böhm M , Ikehara Y , Rieke L
Published: April 2011

Myocardial capillary endothelial cells, arteriolar endothelial cells, and the arterial adventitia show positive alkaline phosphatase (AP) enzyme reaction and immunoreactivity in both rat and human hearts. In guinea pigs, however, capillary endothelial staining is discontinuous and arterial adventitia is negative. The ultrastructural correlate of discontinuous capillary staining is a pronounced labeling of pericytes in guinea pig heart and relatively weak endothelial staining. In rat and human heart, enzyme reaction products are localized mainly on plasma membranes and cytotic vesicles of endothelial cells. Comparison of two strains of rat reveals a more dense deposition of enzyme reaction product along the luminal and particularly along the abluminal plasma membrane of Sprague-Dawley rats than of Wistar rats. Quantitative analysis of immunogold labeled anti-AP antibody density confirms the pronounced polarity of capillary endothelial cell labeling in Sprague-Dawley rats. More than 80% of total endothelial AP protein in Sprague-Dawley rats is localized over the abluminal plasma membrane and basal lamina, as compared with less than 30% in Wistar rats. Moreover, the total endothelial cell labeling is almost sixfold higher in Sprague-Dawley than in Wistar rats. Total endothelial labeling and proportion of labeling on the abluminal endothelial plasma membrane in human hearts is intermediate between the two strains of rat. The strain and species differences in enzyme distribution could provide important information concerning enzyme function.
Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.
Authors: Longin A , Souchier C , Ffrench M , Bryon P A
Published: April 2011

To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.
Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.
Authors: Barka T , van der Noen H
Published: April 2011

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.