bluuebunny/crossref_metadata_2025 huggingface dataset:
Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.
Authors: Lea P , Gross D K
Published: April 2011

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.
[3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.
Authors: Maki A A , Baskin D G , Stahl W L
Published: April 2011

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.
Distribution of chymase-containing mast cells in human bronchi.
Authors: Matin R , Tam E K , Nadel J A , Caughey G H
Published: April 2011

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.
A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.
Authors: Shi S R , Coté C , Kalra K L , Taylor C R , Tandon A K
Published: April 2011

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.
Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.
Authors: Michetti F , Grilli Caiola M , Botti F , Bertini G , Cocchia D
Published: April 2011

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.
An innovative coating technique for light and electron microscopic autoradiography.
Authors: Kornhauser G V , Krum J M , Rosenstein J M
Published: April 2011

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.
Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.
Authors: Phend K D , Weinberg R J , Rustioni A
Published: April 2011

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.
Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies.
Authors: Wooding F B , Morgan G , Forsyth I A , Butcher G , Hutchings A , Billingsley S A , Gluckman P D
Published: April 2011

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.
Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.
Authors: De Vries J E , Kornips F H , Wiegant J , Moerkerk P M , Senden N , Schutte B , Geraedts J P , Bosman F T , Ten Kate J
Published: April 2011

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.
Accurate determination of the time of cell birth using a sequential labeling technique with [3H]-thymidine and bromodeoxyuridine ("window labeling").
Authors: Repka A M , Adler R
Published: April 2011

During tissue embryogenesis, precursor cells divide actively and eventually withdraw from the mitotic cycle before differentiation. Accurate information about the time of terminal mitosis ("birthdate") of precursors is of vital importance for studying relationships between cell proliferation and differentiation. Methods presently available for birthdate determination, based on "pulse" or "cumulative" labeling with either tritiated thymidine (3HT) or bromodeoxyuridine (BrDU) incorporated into DNA during the mitotic cycle, allow only the approximate timing of terminal mitosis. To overcome this limitation, we have developed a "window labeling" technique based on the sequential administration of 3HT and BrDU. Chick retinal precursor cell cultures were first exposed to 3HT and, after a specified time interval, also to BrDU. After 6 days the cultures were fixed and processed for BrDU immunocytochemistry and 3HT autoradiography. Three populations of cells could be easily identified: (a) unlabeled cells, representing post-mitotic cells before label exposure; (b) BrDU-labeled cells [either 3HT (+)/BrDU (+) or 3HT (-)/BrDU (+)], representing those that continue dividing after the addition of BrDU; and (c) "window-labeled" cells, 3HT (+)/BrDU (-), which are those undergoing their last round of DNA synthesis during the interval between 3HT and BrDU administration. Control experiments demonstrated that this method allows birthdate determinations with a resolution of hours or minutes and is essentially free of deleterious effects on precursor cell survival and differentiation.
Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.
Authors: Itoh J , Osamura R Y , Watanabe K
Published: April 2011

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.
Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.
Authors: Le Guellec D , Trembleau A , Pechoux C , Gossard F , Morel G
Published: April 2011

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.
Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin.
Authors: Akimoto Y , Obinata A , Endo H , Hirano H
Published: April 2011

The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane and that various components of the basement membrane appeared at different times during the reconstruction.
[3H]-adenine metabolism and radiation damage during in vitro development of the kidney.
Authors: Salonen J , Saxén L , Lehtonen E , Raivio K O
Published: April 2011

We followed the early post-induction changes in nucleic acid synthesis of the metanephric kidney anlage in vitro. Enhanced incorporation of [3H]-thymidine and [3H]-adenine was detected, but several factors were shown to influence the interpretation of such in vitro experiments. The incorporation is dependent not only on the stage of development of the target organ but also on its transfer to organ culture, as early rudiments require an "adaptive" pre-cultivation to stabilize their metabolism; at more advanced stages growth and DNA synthesis proceed without delay. Another potential artifact is radiation damage which is caused by the incorporated radioisotope and can be detected in prolonged cultures. A [3H]-adenine pulse of more than 1 microCi/ml for 2 hr leads to definite growth retardation, and a 10-microCi/ml pulse causes extensive cell death and atrophy on a 4- to 6-day subculture. The radiation damage is dose-dependent and of variable severity in the different cell lineages within the organ. Since the radioisotope doses were in the range of those commonly used for monitoring cell proliferation and metabolism, we stress the risk of obtaining artifactual results, especially in prolonged cultures after pulse-labeling.
Differential extractability of calcium and pectic substances in different wall regions of epicotyl cells in young flax plants.
Authors: Jauneau A , Morvan C , Lefebvre F , Demarty M , Ripoll C , Thellier M
Published: April 2011

We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.
Novel method for performing carbonic anhydrase histochemistry and immunocytochemistry on cryosections.
Authors: Matsumoto T , Schwartz G J
Published: April 2011

It is difficult to correlate structure with function in the kidney because of the extensive cell heterogeneity. Carbonic anhydrase (CA) is an enzyme that mediates renal acidification and is found predominantly in proximal tubule and collecting duct cells. We modified Hansson's method for histochemically identifying cellular CA activity on PLP-fixed rabbit kidney sections mounted on Millipore filters, and then removed the filters to perform peanut lectin and antibody labeling on the same sections. There was adequate preservation of morphology, and individual cells could be identified with CA activity in the cytosol and specific antibody or lectin labeling on the cell surfaces.
In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.
Authors: Ikeda T , Nomura S , Yamaguchi A , Suda T , Yoshiki S
Published: April 2011

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.
Distribution of type II collagen mRNA in Xenopus embryos visualized by whole-mount in situ hybridization.
Authors: Bieker J J , Yazdani-Buicky M
Published: April 2011

We have developed a whole-mount histochemical method to monitor the distribution of expressed genes within the intact, developing vertebrate embryo. Background problems that result from alkaline phosphatase- or horseradish peroxidase-based stains have been minimized, enabling both early and late stages of Xenopus embryogenesis to be monitored. The feasibility and utility of this non-isotopic method has been demonstrated by using a specific DNA probe to localize Xenopus laevis Type II collagen mRNA expression to areas surrounding the vacuoles of the notochord in Stage 30 embryos. Expression expands by Stage 41/42 to form a visually striking distribution pattern that includes a variety of chondrogenic tissues such as the vertebrae, otocysts, mandible, and periocular region. Although these experiments focused on expression of a structural gene, the high resolution and sensitivity of the method should allow it also to monitor expression of less abundant mRNA products of non-structural genes such as transcription factors, cytoplasmic regulators, and growth factors. In addition, this approach should be a successful tool to probe expression in normal and perturbed embryos not only of amphibians but also of other vertebrates, including avians and mammals.
Differing cell surface distribution of human leukocyte antigen-DR molecules on epidermal Langerhans cells and eccrine duct cells.
Authors: Imayama S , Yashima Y , Hori Y
Published: April 2011

Scanning electron microscopy (SEM) with immunogold labeling was employed to observe the undersurface of the human epidermis after it was split from dermal connective tissue, in an attempt to localize the molecules actually expressed on cell/tissue surfaces. We found that human leukocyte antigen-DR (HLA-DR) molecules were expressed on the surfaces of eccrine duct cells as well as those of epidermal Langerhans cells (LC) in normal skin. HLA-DR molecules, visualized by the deposition of gold particles, were distributed evenly on the LC surface but were present only along the interdigitating borders of the individual duct cells, thus producing a meshwork pattern on the duct surface. Transmission electron microscopy confirmed that the gold particles labeling cell surface HLA-DR molecules were seen only on the portions of duct cell membranes the interdigitated with neighboring duct cells. These findings suggest that the function of HLA-DR molecules may vary with their location and distribution. On the LC surface, the evenly distributed molecules seem to be well suited for promoting "accessory cell" functions. On duct cell surfaces, the HLA-DR molecules present along the intercellular spaces may be involved in trapping various peptide antigens that pass into the sweat gland filtrate and then are reabsorbed by the excretory duct, since these molecules have a highly permissive capacity for binding various peptides.
Histochemical studies of acid and alkaline phosphatases in rat tooth germs with undecalcified resin-embedded specimens.
Authors: Tenorio D , Germain J P , Hughes F J
Published: April 2011

A novel technique for the histochemical demonstration of acid phosphatase (AcPase) and alkaline phosphatase (AkPase) in hard tissues has been proposed. Fresh, unfixed, undecalcified samples of rat tooth germs and surrounding structures were embedded in LR Gold resin at -20 degrees C. Sections of 2 microns were taken and subsequently processed for enzyme histochemistry. AkPase reaction product appeared as strong linear staining outlining cell boundaries and was present in the enamel organ, dental pulp, and osteoblast cells. Tartrate-resistant AcPase staining was seen exclusively in the osteoclasts of developing alveolar bone. Our results demonstrated that the use of unfixed, undecalcified LR Gold resin-embedded specimens for histochemistry is a novel technique which may be of value for certain studies when decalcification of specimens is undesirable. The technique appears to give good preservation of enzyme activity combined with the ability to prepare sections with excellent morphological detail.
Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.
Authors: Nakamura K , Hasler T , Morehead K , Howard R J , Aikawa M
Published: April 2011

Adherence of Plasmodium falciparum-infected RBCs (PRBC) to endothelial cells causes PRBC sequestration in cerebral microvessels and is considered to be a major contributor to the pathogenesis of cerebral malaria. Both CD36 and thrombospondin (TSP) are glycoproteins that mediate PRBC adherence to endothelial cells in vitro. Because they are both expressed on the surface of endothelial cells, they probably contribute to PRBC sequestration and vascular occlusion in vivo. By applying affinity labeling of receptor binding sites with purified ligands, we showed for the first time that both CD36 and TSP can bind independently to the PRBC surface and that the PRBC receptor(s) for CD36 and TSP are localized specifically to the electron-dense knob protrusions of the PRBC surface. These findings may help in efforts to develop a malaria vaccine to prevent cerebral malaria.
Localization of lysosomal acid phosphatase mRNA in mouse tissues.
Authors: Geier C , Kreysing J , Boettcher H , Pohlmann R , von Figura K
Published: April 2011

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.
Localization of a novel integrin of the beta 1 subfamily in human tissues.
Authors: Favre A , Giunta M , Gu Y , Corte G , Grossi C E
Published: April 2011

We have previously described a novel integrin composed of a beta 1-chain non-covalently linked to an alpha-chain which is biochemically different from those known so far (i.e., alpha 1-alpha 7 and alpha v). This molecule has been identified with a monoclonal antibody (MAb) termed 10.1.2 raised against long-term cultured human thymic epithelial cells (TEC). In this study we analyzed the immunohistochemical distribution of this new integrin in a variety of human tissues. MAb 10.1.2 stains several types of endothelial and epithelial cells. Among the endothelia, a strong reaction was detected in the HEV of lymphoid organs including thymus, lymph node, tonsil, and mucosa-associated lymphoid tissue. Epithelial localizations of note were those in the basal layer of the epidermis and of other stratified squamous epithelia, where the lateral and apical but not the deep surfaces of most cells were stained. A variety of water-electrolyte transporting cells in sweat glands, salivary glands, and kidney were also stained at their deep surface. The latter findings suggest that this molecule may subserve other functions in addition to those related to cell adhesion.
Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.
Authors: Hou W Y , Pimenta P F , Shen R L , Da Silva P P
Published: April 2011

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.
Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.
Authors: Price R L , Nakagawa M , Terracio L , Borg T K
Published: April 2011

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.
Intranuclear localization of phospholipids by ultrastructural cytochemistry.
Authors: Maraldi N M , Zini N , Squarzoni S , Del Coco R , Sabatelli P , Manzoli F A
Published: April 2011

The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.
Expression of 20 KD homologous restriction factor of complement on myocardial cells: an immunohistochemical study using acetone-fixed and paraffin-embedded tissues.
Authors: Tada T , Okada H , Okada N , Tateyama H , Eimoto T
Published: April 2011

Expression of 20 KD homologous restriction factor of complement (HRF20) (CD59) in normal human heart was immunohistochemically examined with monoclonal antibody (MAb) 1F5. HRF20 was clearly demonstrated on the cell surface membrane and intercalated discs of myocardial cells throughout the ventricular walls. Therefore, this factor may protect normal cardiomyocytes from complement deposition, which has been demonstrated on infarcted cardiomyocytes. Expression of HRF20 was also observed on endothelial cells of the endocardium and on blood vessels, including arteries, capillaries, and veins, and on the Schwann-cell sheath of peripheral nerve fibers. In the present study, we found that the epitopes of HRF20 were well preserved for immunohistochemistry even in acetone-fixed and paraffin-embedded tissues, as well as in frozen tissues that have been conventionally used for HRF20. This method using paraffin sections allows for easier staining and greater accuracy in histological examination.
Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry.
Authors: Jones H B , Clarke N A , Barrass N C
Published: April 2011

We report modifications to immunocytochemical detection procedures for proliferating cell nuclear antigen (PCNA) which permit its identification in liver samples previously fixed for BrdU immunocytochemistry. Both methods have been used for the assessment of phenobarbital-induced cell proliferation in rat liver. The difficulties associated with the hitherto unsuccessful application of PCNA immunocytochemical methods to tissues fixed in formalin for BrdU visualization were overcome by epitope unmasking with acid hydrolysis, extension of primary antiserum (PC10) incubation, and employment of streptavidin-ABC-HRP. BrdU delivery via osmotic minipumps for 48 hr before euthanasia, followed by fixation in cold formalin for 14 days, yielded reliable and reproducible hepatocellular labeling and a peak of cell proliferation in all lobes on Day 3 (i.e., labeling during Days 1-3) of dosing with 80 mg/kg/day phenobarbital. Labeling indices (LI) of both control and phenobarbital-treated liver were lower in the left and right median lobes as compared with the lateral lobes. In sections of the left lateral lobe from the same liver, PCNA immunocytochemistry revealed a peak of proliferative activity (about one third of the maximum LI generated by BrdU incorporation) on Day 1. These findings, together with the advantages and disadvantages of both techniques, are discussed in the context of their applications to different investigative requirements.
Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells.
Authors: Poulsen M D , Hansen G H , Dabelsteen E , Høyer P E , Norén O , Sjöström H
Published: April 2011

Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein, since the gold particles appear all over the granules and are not specifically associated with the granule membrane.
Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and influenza virus.
Authors: Sakai K , Kawaguchi Y , Kishino Y , Kido H
Published: April 2011

The intracellular localization in rat bronchiolar epithelial cells of a novel trypsin-like protease named tryptase Clara, a possible activator of inactive viral fusion glycoprotein of influenza A and wild-type Sendai virus in the respiratory tract, was examined by electron microscopy. In thin sections embedded in LR White, gold particles indicating immunoreactivity of tryptase Clara were detected specifically in secretory granules of Clara cells. No immunoreactivity was detected in bronchiolar ciliated cells, alveolar cells including epithelial Type I and II cells, or alveolar macrophages. Some granules enveloped in a thin membrane and labeled intensely with immunogold particles were seen protruding from peripheral and submembrane regions and a few were observed free in the airway lumen. Scanning electron microscopy also revealed smooth droplets along the main body of Clara cells. These data suggest that tryptase Clara is secreted into the bronchiolar lumen. These findings are the first to show the subcellular localization of tryptase Clara in rat bronchioles and suggest the site of proteolytic activation of the progeny of enveloped pneumotropic viruses, such as Sendai virus and influenza virus.
Densities of NADPH-ferrihemoprotein reductase and cytochrome P-450 molecules in the endoplasmic reticulum membrane of rat hepatocytes.
Authors: Watanabe J , Asaka Y , Fujimoto S , Kanamura S
Published: April 2011

In hepatocytes, NADPH-ferrihemoprotein reductase (reductase) has been hypothesized to exist as aggregates or micelles in endoplasmic reticulum (ER) membrane. However, if the number of reductase molecules per unit area of ER is low, this hypothesis cannot explain how a few reductase molecules efficiently reduce many P-450 molecules. To test this hypothesis, we estimated the numbers of reductase and P-450 molecules per unit ER area (reductase and P-450 densities) by microphotometry of the two enzymes in conjunction with morphometry of ER in periportal, midzonal, and perivenular rat hepatocytes. The reductase density in periportal, midzonal, and perivenular hepatocytes (107-179 molecules/microns 2 of ER) was high enough to efficiently reduce all P-450 molecules in the ER, although the value in perivenular hepatocytes was lowest owing to the relatively greater amount of ER in this region. The pattern of sublobular gradient in the reductase density was similar to that in the P-450 density. Consequently, the molar ratio of P-450 to reductase in ER was similar (about 40:1) in hepatocytes regardless of their positions within the liver lobule.
Ultrasound-amplified immunohistochemistry.
Authors: Podkletnova I , Alho H
Published: April 2011

We describe a novel technique for improving the sensitivity of immunofluorescence staining by use of ultrasonic irradiation. Free-floating vibratome sections from rat cerebellum were incubated with primary antiserum and simultaneously were briefly exposed to ultrasound (US) in a conventional ultrasound bath. After the US treatment, a conventional immunohistochemical method was employed. Two different antisera and two conventional immunohistochemical detection systems were tested. In all cases a 10-20-sec US treatment strengthened immunoreactivity considerably. Irradiated samples showed a good morphology compared with non-irradiated sections. Our results demonstrate that with ultrasonic treatment the dilutions of primary antibodies can be increased and the incubation times of primary antisera can be reduced. The ultrasonic method described here requires no special equipment. It is easy, reproducible, and it can be considered a new method for the enhancement at immunohisto- and cytochemical staining of free-floating vibratome sections.
Secretion of glandular kallikrein and renin from the basolateral pole of mouse submandibular duct cells: an immunocytochemical study.
Authors: Penschow J D , Coghlan J P
Published: April 2011

Glandular kallikrein from salivary glands in rats has been measured in the circulation and has been shown to have local vasoactive effects. In mice, renin and epidermal growth factor from the submandibular gland (SM) also reach the circulation, as both have been measured in plasma. The route by which these peptides enter the blood from their site of synthesis in ducts of the SM is unclear. We have investigated by immunocytochemistry the secretory pathways for kallikrein and renin from salivary duct cells in mice. The renal/pancreatic kallikrein-secreting cells of the striated and excretory ducts of the SM were distinguished from the granular convoluted tubule (GCT) cells which secrete other glandular kallikreins on the basis of data obtained in previous studies, in which we used gene-specific oligonucleotide probes to identify the expressing cell types. Renal/pancreatic kallikrein was apparently secreted constitutively from the basolateral surface of striated duct cells and in secretory vesicles from excretory duct cells, whereas apical secretion occurred via the regulated pathway in both cell types. Glandular kallikreins and renin synthesized in GCT cells were secreted from the basolateral surface by dissolution of granules at the cell membrane. There were fenestrated capillaries underlying the duct tree which would enable the secreted products to reach the circulation.
The immunohistochemical heterogeneity of atheroma macrophages: comparison with lymphoid tissues suggests that recently blood-derived macrophages can be distinguished from longer-resident cells.
Authors: Poston R N , Hussain I F
Published: April 2011

We studied the antigenic markers of macrophages (Mphs) in atherosclerotic human arteries by immunohistochemistry and compared them with the patterns in Mph subpopulations of tonsil and lymph node, which are also described. The staining of atheroma intimal Mphs was assessed semiquantitatively in the subendothelial, mid, and outer intima. Three patterns of reactivity with Mph antibodies were recognized. (a) Pan-Mph (antibodies HAM56, EBM11, and CD14 group). Staining was maximal in the mid-intimal zone. (b) Subendothelial Mphs (anti-muramidase, anti-alpha-1-antitrypsin and MAC387). In lymphoid tissue, sinusoidal Mphs and a few inflammatory Mphs were stained, as well as blood monocytes. This group of antibodies recognizes Mphs that are likely to be recently blood-derived (RBD-Mphs). (c) Antibodies reactive with various histiocyte populations in lymphoid tissues (anti-Factor XIII; anti-HLA Class II and LN2) also gave maximal staining in the mid-intimal zone, but differences between lesion types suggest that they are recognizing heterogeneous subpopulations of Mphs. These observations demonstrate the heterogeneity of tissue Mphs and suggest that an insight into the dynamics of tissue Mphs can be obtained from the cell phenotype. They indicate that all stages of atherosclerosis can have an outward traffic of Mphs from the blood through the arterial intima.