bluuebunny/crossref_metadata_2025 huggingface dataset:
Charcot-Leyden crystal protein distribution in basophils and its absence in mast cells that differentiate from human umbilical cord blood precursor cells cultured in murine fibroblast culture supernatants or in recombinant human c-kit ligand.
Authors: Dvorak A M , Ishizaka T , Letourneau L , Albee E A , Mitsui H , Ackerman S J
Published: April 2011

Suspension cultures of human umbilical cord blood mononuclear cells supplemented with c-kit ligand-containing additives give rise to a mixture of cells belonging to several lineages. Among those that differentiate in quantity are mature basophils, immature mast cells, and neutrophilic myelocytes. We used an ultrastructural immunogold method to detect the Charcot-Leyden crystal (CLC) protein, an eosinophil- and basophil-specific protein, to study cells that were obtained at sequential times from 3 to 14 weeks in culture. Basophils (and eosinophils, which were present in smaller numbers) labeled for the CLC protein; mast cells did not. The labeled basophil subcellular sites included formed intragranular, cytoplasmic and nuclear CLCs, cytoplasmic particle-filled and homogeneously dense granules, cytoplasm, nucleus, plasma membrane, and cytoplasmic and Golgi area vesicles. Individual basophil ultrastructural phenotypes similar to those associated with stimulated release and recovery reactions showed the expected variations in the gold-labeled subcellular compartments. Macrophages also were labeled for CLC protein within endocytotic-lysosomal structures; neutrophilic myelocytes did not contain CLC protein. On the basis of findings reported here, the combined ultrastructural morphology and immunogold phenotyping of cells differentiating in c-kit ligand-supplemented cultures allows accurate lineage assignment of the developing cells.
Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.
Authors: De Boer W I , Rebel J M , Vermey M , Thijssen C D , Van der Kwast T H
Published: April 2011

The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes.
Three-dimensional co-localization of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.
Authors: Ploton D , Gilbert N , Ménager M , Kaplan H , Adnet J J
Published: April 2011

Silver dots deposited specifically on proteins of the nucleolar organizer regions (Ag-NOR proteins) after a one-step silver staining technique were visualized in cells in culture, in cells in smears, and in tissue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin A3, DNA and silver dots could be observed either individually or simultaneously. Therefore, it was possible to study the three-dimensional organization of nucleolar silver-stained structures relative to DNA with a high X, Y, and Z resolution. Our results showed that the argyrophilic components are organized as a twisted necklace structure within interphase nucleoli of cells in culture. We also demonstrated a striking three-dimensional symmetric disposition of NORs within the two sets of chromosomes in telophase cells. Similar results were obtained for cells in smears, although their three-dimensional organization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous demonstration in non-confocal transmitted light, together with that of DNA in confocal mode, appeared very useful to study their localization within nuclei and mitotic chromosomes.
Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy.
Authors: Leger I , Robert-Nicoud M , Brugal G
Published: April 2011

The recent application of DNA cloning and non-radioactive in situ hybridization techniques has strengthened the hypothesis of an ordered chromatin structure in interphase nuclei. The arrangement of specific chromosomal regions is not random and is strongly suspected to vary with functional activity. The combination of in situ hybridization and immunocytochemistry, allowing simultaneous detection of nucleic acid sequences and specific antigens in the same nucleus, has already made significant contributions to the study of gene expression, to simultaneous karyotyping and phenotyping of tumor cells, and to in situ analysis of viral infections. This report emphasizes the considerable interest of such combined techniques for functional in situ mapping of the genome at the individual cell level. We propose a method that combines fluorescence immunocytochemical detection of nucleolar proteins and fluorescence in situ hybridization of centromeric and telomeric probes specific for chromosome 1 in two cultured human cell lines. The preparative constraints for a broad application of this procedure are defined so that the cell preparations can be further analyzed by fluorescence microscopic imaging techniques and confocal laser scan microscopy. The two selected sequences of the human chromosome 1 can be localized in the nucleus with respect to nucleolar proteins in a one-step fluorescence microscopic observation.
Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae.
Authors: Parton R G
Published: April 2011

The ultrastructural distribution of the ganglioside GM1 was investigated in A431 cells. After fixation, the cells were frozen in liquid nitrogen, freeze-substituted, and then embedded in Lowicryl resin at -45 degrees C. By use of the cholera toxin-binding subunit adsorbed to gold as a specific probe to label on the sections, GM1 was shown to be present in endocytic organelles, in the trans-Golgi network, and on the plasma membrane, but was not detectable in the endoplasmic reticulum. GM1 was not distributed uniformly over the plasma membrane but was concentrated approximately four-fold in non-coated invaginations. These were identified as caveolae by labeling frozen sections of cholera toxin-gold surface-labeled cells with antibodies to VIP-21/caveolin. The results strengthen the functional analogy between caveolae and sorting domains of the TGN in polarized epithelial cells.
Digital confocal microscopy allows measurement and three-dimensional multiple spectral reconstruction of Neisseria gonorrhoeae/epithelial cell interactions in the human fallopian tube organ culture model.
Authors: Gorby G L
Published: April 2011

A strategy for measuring Neisseria gonorrhoeae attachment and invasion in the human Fallopian tube organ culture (FTOC) model via computerized image analysis (CIA) combined with "digital" confocal microscopy (DCM) was tested. DCM on serial image stacks of fluorescent latex beads reduced out-of-focus light propagation in the Z-axis (p < 0.005) and improved the shape factor of lateral three-dimensional reconstructions of the beads (p < 0.001). Sections of tissue infected for 44 hr with piliated, Opa+ gonococci were stained with fluorescein-labeled monoclonal anti-gonococcal antibodies, rhodamine-labeled phalloidin, and Hoechst 33342. Serial images collected at identical focal planes for each fluorochrome were subjected to DCM. Epithelial cytoplasmic regions of interest defined by rhodamine-stained actin were superimposed on the corresponding fluorescein-stained and Hoechst-stained images. Fluorescent objects defined by gray-scale threshold were measured by computerized image analysis using different border treatments to differentiate attached from intracellular gonococci or count cell nuclei. Compared with raw images, measurement of DCM images was less dependent on threshold choice (p < 0.05). DCM augments conventional microscopy in removing out-of-focus light from fluorescent images, in reconstructing three-dimensional images, and in quantitatively differentiating extracellular from intracellular gonococci in a natural target tissue.
A combined ELISA-immunoelectron microscopic approach for topological mapping of membrane protein epitopes: application to the nicotinic acetylcholine receptor.
Authors: Schmutz M , Kling D , Tzartos S , Brisson A
Published: April 2011

Identification of epitope localization on either side of the lipid membrane by immunoelectron microscopy constitutes an intrinsic powerful method of structure determination for membrane proteins. We have developed a method allowing measurement and observation, under almost identical experimental conditions, of the binding of monoclonal antibodies (MAb) to membrane-bound acetylcholine receptor from Torpedo marmorata electric tissue. This method, based on ELISA and electron microscopy of negatively stained specimens, was developed with MAb of known epitope specificity. With native membrane fragments, we found that MAb bound to extracellular epitopes in a stoichiometric manner, whereas almost no binding was detected for intracellular epitopes. The treatment based on tissue homogenization in the presence of Zn2+ ions and sucrose resulted in the formation of large, stable openings, rendering accessible about 25% of intracellular epitopes. Electron microscopic observations showed a clear distinction between antibody binding to either intracellular or extracellular epitopes, both with native and Zn(2+)-treated membranes. In addition, the binding of one antibody directed against an extracellular epitope was strikingly dependent on the packing density of acetylcholine receptor molecules, thus enabling us to further distinguish between two levels of accessibility for extracellular epitopes. The method presented here is of general application for studies of epitope mapping in membrane proteins.
Golgi-Colonnier method: correlation of the degree of chromium reduction and pH change with quality of staining.
Authors: Angulo A , Merchán J A , Molina M
Published: April 2011

We examined the role of chromium reduction in the Golgi-Colonnier method, correlating the quality of neuronal impregnation with the levels of hexavalent (CrVI) and trivalent (CrIII) chromium in the tissue and in the chromation fluid (CF). The concentrations of both chromium species were assessed by measuring spectrophotometrically the CrVI before and after oxidizing the sample and by calculating the ratio of CrVI to total chromium (chromium ratio, CrR). The CrR was almost identical in the tissue and the CF, decreasing exponentially during chromation due to a progressive consumption of CrVI to form CrIII. Satisfactory cell impregnation was obtained only when the CrR was 0.45-0.7, regardless of other factors. The CrR values could be accurately predicted by the pH increase of the CF; this increase has proven to be a most reliable criterion to decide the endpoint of the chromation process. The dependence of cell staining on the [CrIII], together with the well-known ability of this species to bridge proteins, suggests that the key event for cell impregnation is the cross-linking of neuronal proteins by CrIII polymers.
New monoclonal antibodies directed against mouse follicular dendritic cells.
Authors: Imazeki N , Takeuchi A , Senoo A , Fuse Y
Published: April 2011

The follicular dendritic cells (FDC) in B-lymphoid follicles are unique reticular cells that retain immune complexes on their surfaces. We developed new monoclonal antibodies (MAb), SKY01, 28, 41, and 49, against mouse FDC without useful cell markers. Immunohistochemical study of spleen and lymph node tissues from Balb/c, C3H, and C57BL/6 mice revealed that SKY01 and 49 were highly specific for FDC, whereas SKY28 and 41 reacted with other stromal components as well as FDC. On immunoelectron microscopy (IEM), reaction products for all MAb were localized on the surfaces of FDC. In ontogenetic study of Balb/c spleen, FDC precursors were not immunodetected with any of the MAb at 1 week after birth. The FDC that first appeared as immune complex-retaining cells at 2 weeks were a subpopulation of SKY01-, 49-, 28+, and 41+ reticular cells. At 3 weeks, FDC were positive for all MAb, like adult spleen. These results indicate that our MAb recognize differentiation antigens of FDC. Comparative immunohistochemical studies of spleen from athymic nude and severe combined immunodeficiency mice suggested that B-cells may be required for the differentiation of FDC. Consequently, these MAb are considered useful tools for research on FDC.
A protein-specific monoclonal antibody to rat liver beta 1-->4 galactosyltransferase and its application to immunohistochemistry.
Authors: Kawano J , Ide S , Oinuma T , Suganuma T
Published: April 2011

We have produced a new protein-specific monoclonal antibody (MAb) to rat liver beta 1-->4 galactosyltransferase. This MAb, GTL2, was selected as the most reactive IgG to a periodate-treated antigen. Antigen and protein specificities of GTL2 were verified by immunoblotting of a non-glycosylated recombinant protein of human galactosyltransferase and enzymatically deglycosylated rat galactosyltransferase. Using GTL2, an immunohistochemical study was done in rat liver, epididymis, and salivary glands. Intense staining was observed in Golgi areas of epididymal duct epithelial cells, and submandibular and sublingual acinar cells. Hepatocytes showed weaker staining. Immunoelectron microscopic observation revealed that the staining was exclusively localized in trans-Golgi membranes of these cells.
A comparison of heating modes in rapid fixation techniques for electron microscopy.
Authors: Leonard J B , Shepardson S P
Published: April 2011

Recent studies have established that microwave irradiation during aldehyde fixation of electron microscopy specimens can reduce fixation times substantially. Similar reductions in duration of histochemical and light microscopy procedures have been reported. Both thermal and non-thermal effects of microwaves have been proposed to explain these dramatic decreases in processing time. Possible thermal effects include increases in fixative diffusion and reaction rates and increased formation of glutaraldehyde monomers. Proposed non-thermal effects include preferential orientation of fixative molecules by the microwave field and other more speculative direct microwave effects. Several reported attempts to produce rapid fixation without temperature increase by cooling specimens during irradiation have produced conflicting results. If rapid fixation is a thermal effect, other heating modes in addition to microwave exposure should produce similar effects. We show that for mouse liver samples (< or = 1 mm3) comparable fixation can be obtained with microwave irradiation, conductive and convective heating in a waterbath, and resistive heating with a low-frequency (1 kHz) current passed through the fixative solution. We also show that using an efficient convective cooling method to prevent temperature increase during microwave exposure produces unsatisfactory fixation. These results are consistent with thermal mechanisms for rapid fixation.
Immunolocalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies: evidence of a proteinase specifically expressed in keratinizing squamous epithelia.
Authors: Sondell B , Thornell L E , Stigbrand T , Egelrud T
Published: April 2011

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered serine proteinase, which has been purified from human plantar stratum corneum. Evidence has been presented that it may play a role in the terminal stages of epidermal turnover, especially in desquamation. Two mouse monoclonal antibodies (MAb) were raised, TE4b and TE9b, that reacted specifically with SCCE in immunoprecipitation, immunoblotting, and gel-exclusion chromatography. When used in immunohistochemical experiments with the peroxidase-anti-peroxidase method, both MAb detected an antigen located in high suprabasal keratinocytes of the epidermis in normal human skin and at the vermilion border of the lip, with maximal staining of the stratum granulosum. In the hair follicles the MAb reacted with the inner root sheet only. In human oral mucosa the MAb stained the high suprabasal epithelial cells of the hard palate. This is a site where the epithelium forms an orthokeratotic stratum corneum. There was no specific staining of the epithelium of the lip mucosa or the buccal mucosa, where the epithelium does not form a stratum corneum under non-pathological conditions. A correlation therefore seems to exist between the presence of SCCE in high suprabasal cells and the ability of the epithelium to form an orthokeratotic cornified layer. We suggest that SCCE is specifically expressed in keratinizing squamous epithelia and that its expression may be part of the terminal differentiation program of this type of epithelium. These results also give further support to the idea that SCCE may play a role in the turnover and/or formation of the stratum corneum.
A comparison of techniques for localizing actin and tubulin in hyphae of Saprolegnia ferax.
Authors: Kaminskyj S G , Heath I B
Published: April 2011

We have evaluated protocols for immunofluorescence (IF) staining of the potentially interacting actin filaments (F-actin) and microtubules in hyphae of Saprolegnia ferax, using rhodamine-phalloidin (RP) and freeze-substitution electron microscopy (FSEM), respectively, as standards for their distribution. Saprolegnia has four distinguishable cortical F-actin populations with characteristic organizations and RP- and actin-IF-staining affinities, all of which could be labeled with both probes after some protocols. Other protocols stained only some of the populations. Cortical F-actin was always more reproducibly and sharply stained with RP than IF, indicating that the former is the probe of choice for F-actin in these cells. Although no single IF protocol revealed all of the F-actin and microtubule populations, showing the potential need to optimize protocols for specific antibodies, simultaneous localization was readily achieved by dual labeling with RP and tubulin IF. Tubulin IF patterns differed from FSEM: mitotic spindles were revealed but not the more abundant prophase microtubule arrays, and the cytoplasmic microtubules were subapically displaced and bundled into long cables. These cables, which apparently linked nuclei, indicate a previously undetected involvement in nuclear spacing. The tubulin antibody successfully used for IF failed to recognize any proteins in immunoblots, indicating that immunoblots may not always be a useful indicator of success with IF.
Monoclonal antibodies to a zinc-binding protein of rat Paneth cells.
Authors: Sawada M , Horiguchi Y , Abujiang P , Miyake N , Kitamura Y , Midorikawa O , Hiai H
Published: April 2011

Paneth cells are morphologically well characterized but their function has been not elucidated. Previously, we identified and purified a 90 KD zinc-binding protein (ZBPP-1) in rat intestine that was localized to Paneth cell granules, consistent with their high zinc content. To further elucidate the structure and function of ZBPP-1, we immunized Balb/c mice with purified ZBPP-1 and identified four independent monoclonal antibodies (MAb) producing MAb ZIP-1 (IgM), ZIP-2 (IgG1), ZIP-3 (IgM), and ZIP-4 (IgM). Immunohistochemistry (IHC) and immunoelectron microscopy (IEM) with these MAb showed positive staining of Paneth cell cytoplasmic granules. MAb ZBPP-1 also stained a population of mononuclear cells in the lamina propria of digestive tract mucosa and a few cells in spleen, presumably a subset of macrophages. These MAb will provide a useful tool to study the function of Paneth cells in human health and disease, since they cross-reacted with human intestinal Paneth cells and mucosal mononuclear cells.
Basement membrane-specific chondroitin sulfate proteoglycan is abnormally associated with the glomerular capillary basement membrane of diabetic rats.
Authors: McCarthy K J , Abrahamson D R , Bynum K R , St John P L , Couchman J R
Published: April 2011

We have previously reported the production of monoclonal antibodies (MAb) recognizing the core protein of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG). Using immunohistochemical techniques, we have shown that BM-CSPG is present in almost every basement membrane, one exception being the normal glomerular capillary basement membrane (GBM), where it is absent. In the present study of mature kidneys we examined the distribution of BM-CSPG in streptozocin-induced diabetes mellitus in rats. We found BM-CSPG atypically associated with the GBM of diabetic animals as early as 1 month after induction of diabetes mellitus. Immunoelectron microscopy (IEM) of affected capillary loops showed BM-CSPG present in the subendothelial matrix in areas of GBM thickening and absent in areas where the GBM appears to be of normal thickness. Moreover, the association of BM-CSPG with regions of the pericapillary GBM affects the morphology of the capillary endothelial cells within these areas, directly displacing the cell body from the GBM proper and causing loss of fenestrae. These new data on BM-CSPG distribution reflect abnormal glomerular extracellular matrix protein biosynthesis/turnover in diabetes and suggest that BM-CSPG in the GBM might in turn affect normal capillary structure and/or function.
Cytochemical characterization of sulfo- and sialoglycoconjugates of human laryngeal glandular cells.
Authors: Castells M T , Madrid J F , Avilés M , Martínez-Menárguez J A , Ballesta J
Published: April 2011

The composition and distribution of sulfo- and sialoglycoconjugates in human laryngeal glands have been investigated at light and electron microscopic levels by use of peroxidase-, digoxigenin-, and colloidal gold-conjugated lectins in combination with several chemical and enzymatic deglycosylation procedures. The present study reveals a variety of terminal oligosaccharide sequences in serous and mucous glands. Serous cells contained glycoconjugates with terminal Neu5Ac (alpha 2-3) Gal (beta 1-4) GlcNAc, Neu5Ac (alpha 2-6) Gal/GalNAc, Neu5Ac (alpha 2-3/6) Gal (beta 1-3 GalNAc, GlcNAc, and Gal (beta 1-4) GlcNAc sequences. Scarce SO4Gal(beta 1-3)GalNAc terminal oligosaccharide chans were detected. Serous cells show wide morphological variability of secretory granules (electron lucent, electron dense, and bizonal) with different lectin affinities. Glycoconjugates in human laryngeal mucous glands contained a variety of terminal oligosaccharide sequences including SO4Gal(beta 1-4)GlcNAc, SO4Gal(beta 1-3) GalNAc, SO4GalNAc, and Neu5Ac(alpha 2-3)GalNAc.
Giemsa as a fluorescent stain for mineralized bone.
Authors: Bradbeer J N , Riminucci M , Bianco P
Published: April 2011

We present evidence for a previously unrecognized differential staining effect of Giemsa solution in fluorescence microscopy. The effect consists of selective fluorescent staining of mineralized bone (and elastic fibers) in tissue sections and, like the classical Romanowsky effect, is based on the differential binding of Eosin Y to tissue structures in the presence of Azur II and Methylene Blue. This effect opens the way to new applications of the Giemsa solution in fluorescence microscopy and in confocal fluorescence microscopy.
Three-dimensional relationships between tumor cells and microcirculation with double cyanine immunolabeling, laser scanning confocal microscopy, and computer-assisted reconstruction: an alternative to cast corrosion preparations.
Authors: Rummelt V , Gardner L M , Folberg R , Beck S , Knosp B , Moninger T O , Moore K C
Published: April 2011

The morphology of the microcirculation of uveal melanomas is a reliable market of tumor progression. Scanning electron microscopy of cast corrosion preparations can generate three-dimensional views of these vascular patterns, but this technique sacrifices the tumor parenchyma. Formalin-fixed wet tissue sections 100-150 microns thick from uveal melanomas were stained with the lectin Ulex europaeus agglutinin I (UEAI) and proliferating cell nuclear antigen (PCNA) to demonstrate simultaneously the tumor blood vessels and proliferating tumor cells. Indocarbocyanine (Cy3) was used as a fluorophore for UEAI and indodicarbocyanine (Cy5) was used for PCNA. Double labeled sections were examined with a laser scanning confocal microscope. Images of both stains were digitized at the same 5-microns intervals and each of the two images per interval was combined digitally to form one image. These combined images were visualized through voxel processing to study the relationship between melanoma cells expressing PCNA and various microcirculatory patterns. This technique produces images comparable to scanning electron microscopy of cast corrosion preparations while permitting simultaneous localization of melanoma cells expressing PCNA. The microcirculatory tree can be viewed from any perspective and the relationship between tumor cells and the tumor blood vessels can be studied concurrently in three dimensions. This technique is an alternative to cast corrosion preparations.
Localization of an ecto-ATPase/cell-CAM 105 (C-CAM) in the rat parotid and submandibular glands.
Authors: Murphy H C , Hand A R , Dowd F J
Published: April 2011

We investigated an ecto-ATPase/cell-CAM 105 (C-CAM), previously shown to be distinct from the Ca2+ pump, in rat parotid and submandibular glands. Polyclonal antibodies raised against the enzyme were employed using indirect immunofluorescence, peroxidase-anti-peroxidase (PAP), and electron microscopic immunogold labeling procedures to visualize the location of the enzyme. In the PAP-stained sections and with immunofluorescence, labeling was observed on the luminal and lateral surfaces and the intercellular canaliculi of the acinar cells of both glands. The luminal surface of the intercalated ducts was brightly stained, whereas those of the striated and excretory ducts were less prominently labeled. The basal surface of the acinar cells in the parotid gland and the lateral and basal surfaces of the duct cells were not labeled. Apparent labeling was observed on the basal surface of the submandibular acinar cells. Electron microscopy revealed that for both glands the enzyme was primarily localized along the luminal border of the acinar cells, mainly in association with microvilli, with slightly less reactivity along the intercellular canaliculi and lateral borders and relatively little along the basolateral membranes. Gold labeling was also observed on the luminal borders of the intercalated and striated ducts. Possible functions of the C-CAM include breakdown of ATP, stabilization of the microvillar membranes, cell adhesion, and involvement in secretory mechanisms.
Zonal heterogeneity of calcium distribution in rat hepatocytes: an electron microscopic study with a combined glutaraldehyde-osmium-pyroantimonate technique.
Authors: Angermüller S , Juchem R , Beier K , Konrad T , Kusterer K
Published: April 2011

Potassium pyroantimonate in combination with osmium tetroxide is an excellent technique for investigation of the subcellular distribution of calcium ions in glutaraldehyde-fixed liver tissue. Under appropriate incubation conditions potassium ions are replaced by calcium ions to form an insoluble electron-dense calcium antimonate precipitate. The presence of calcium within this antimonate precipitate is confirmed by the electron spectroscopic imaging (ESI) technique. Chelator treatment with EGTA [ethylene glycol-bis-(beta-amino-ethyl ether) N',N',N'-tetraacetic acid] is usually used as a control, preventing the precipitation of calcium. In our investigation, computerized image analysis revealed a gradient of the calcium distribution from the periportal to the pericentral area. A finely dispersed precipitate was found in the mitochondrial matrix and in the euchromatin of the nuclei of the periportal hepatocytes, and in the organelles of the pericentral area a coarser deposit in lower concentration was recognized. Our findings indicate that periportal and pericentral hepatocytes retain their zonal characteristics also with respect to calcium concentration in mitochondria and nuclei.
Use of quantitative image microfluorometry to follow fluorescent ricin internalization in single living cells.
Authors: Frénoy J P , Emmanuel F , Salmon J M
Published: April 2011

We used microspectrofluorometry and videomicrofluorometry to follow the binding and internalization of fluorescein-labeled toxic lectin ricin in living Zajdela hepatoma cells. Microspectrofluorometry showed that when ricin was specifically labeled on its B-chain with one molecule of fluorescein (ABF), its fluorescence spectrum did not alter during its binding to the cell surface and subsequent internalization. This enabled us to use image analysis to follow cell internalization of labeled ricin. Accordingly, we measured the appropriate fluorescent cell parameters, comprising total fluorescence intensity, cell surface area, mean fluorescence intensity and its standard error, and used the measurements for mono- and biparametric studies of cell fluorescence distribution. The results showed that (a) ricin binds two different subpopulations of Zajdela hepatoma cells, (b) Zajdela hepatoma cells internalize ricin rapidly and after a relatively stable period of 1-2 hr, internalization starts again at 4 hr, and (c) the distribution of intracellular fluorescence is heterogeneous and ABF accumulates in certain cellular localizations. Our results demonstrate that quantitative microfluorometry is an effective and interesting approach for real-time studies of macromolecule internalization in living cells.
Morphometric studies of the localization of the glucocorticoid receptor in mammalian cells and of glucocorticoid hormone-induced effects.
Authors: Akner G , Wikström A C , Mossberg K , Sundqvist K G , Gustafsson J A
Published: April 2011

We studied the subcellular distribution of the glucocorticoid receptor (GR) by light microscopy (LM) and confocal laser scanning microscopy (CLSM) in different mammalian cell types. The effect of added glucocorticoid hormones on GR distribution was investigated by photometric quantitation on optical sections obtained by CLSM followed by statistical analysis. In the control interphase cytoplasm, the distribution of GR was fibrillar in some and diffuse in other cell types. Fibrillar GR was distributed along cytoplasmic microtubules (MTs) with predilection for a subset of MTs. GR was also observed in the centrosomes. Nuclear GR was both diffuse and granular in distribution. During cell division, GR appeared in the mitotic apparatus at all stages of mitosis. These findings were not fixation-dependent. Glucocorticoid treatment increased both the nuclear and cytoplasmic GR signal. However, this was detectable only after precipitating but not cross-linking fixation. There was both intra- and intercellular GR heterogeneity in the absence and presence of hormone but no indication of a hormone-induced nuclear translocation of GR. We present a hypothetical model of two independent GR populations in the nucleus and cytoplasm, respectively, without any discernible ligand-induced nuclear translocation of GR. The extranuclear GR population may exert effect(s) on site in the cytoplasm without involving nuclear genomic transcription.
Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.
Authors: Shimizu H , Masunaga T , Ishiko A , Hashimoto T , Garrod D R , Shida H , Nishikawa T
Published: April 2011

In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.
A simple, rapid method for isolating RNA from paraffin-embedded tissues for reverse transcription-polymerase chain reaction (RT-PCR).
Authors: Mies C
Published: April 2011

Described here is a simple, rapid technique for isolating RNA from archived formalin-fixed, paraffin-embedded tissues (PETs). Using this modified acid guanidinium thiocyanate method, RNA sufficient as a template for the reverse transcription-polymerase chain reaction (RT-PCR) can be isolated in 2 hr from a single 20-microns-thick section of tissue. Spliced mRNA corresponding to portions of the estrogen receptor (ER) gene was successfully amplified from fixed, embedded human breast cancers containing increased amounts of ER protein. This method makes it feasible to safely and efficiently isolate RNA from large numbers of routinely processed paraffin blocks for retrospective studies of endogenous proteins such as hormone receptors, oncogenes, and viruses.
Glutamate immunoreactivity in rat cerebral cortex is reversibly abolished by 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of phosphate-activated glutaminase.
Authors: Conti F , Minelli A
Published: April 2011

Glutamate (Glu) immunocytochemistry has been widely used to identify presumed gluergic neurons and synapses, but several problems related to the fact that Glu is both a synaptic transmitter and a compound used for metabolic purposes are still unsolved. One of these concerns the intense perikaryal staining observed in perfusion-fixed tissue. Phosphate-activated glutaminase, a key enzyme for the synthesis of releasable glutamate, is inhibited by the diazoketone 6-diazo-5-oxo-L-norleucine (DON), which greatly reduces glutamate release. In the present experiments, DON was either injected intraparenchymally or applied epipially to the sensorimotor cortex of adult Sprague-Dawley rats at concentrations of 0.25-1 mM. Both intraparenchymal and epipial applications of the chemical abolished Glu immunoreactivity in neuron perikarya. Adjacent sections processed for cytochrome oxidase histochemistry, for aspartate immunoreactivity, or stained with thionine showed no changes. The effects of DON application are reversible, as shown in a second series of experiments in which, after 30 min of DON application, animals were allowed to survive for 5-10 days. In these cases, Glu immunoreactivity in cortical neurons was identical to that observed in normal untreated animals. The results reported here suggest that Glu immunoreactivity demonstrated by the present procedure in neuron perikarya is mainly due to Glu produced via phosphate-activated glutaminase.
Processing of the LH/CG receptor and bound hormone in rat luteal cells after hCG-induced down-regulation as studied by a double immunofluorescence technique in conjunction with confocal laser scanning microscopy.
Authors: Lakkakorpi J T , Yang M , Rajaniemi H J
Published: April 2011

We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.
Expression of mRNA for IL1 beta, IL6 and TGF beta 1 in developing human bone and cartilage.
Authors: Dodds R A , Merry K , Littlewood A , Gowen M
Published: April 2011

Using in situ hybridization, we investigated the expression of mRNA for interleukin-1 beta (IL1 beta), interleukin-6 (IL6), and transforming growth factor-beta-1 (TGF beta 1) in sections of developing bone in human osteophytes. The expression was related to the cellular activity of alkaline phosphatase to aid in the identification of pre-osteoblast populations. IL1 beta mRNA was localized in active osteoblasts within distinct areas of intramembranous ossification. However, the expression was sporadic and appeared to occur at a specific stage of the osteoblast life cycle. There was no IL1 beta mRNA expression in any cell types during endochondral ossification. IL6 mRNA expression was located within pre-osteoblasts and in newly differentiated and matrix-secreting osteoblasts; expression was absent or reduced in flattened, inactive osteoblasts. Weak or no IL6 expression was observed in chondroblasts and chondrocytes, respectively. However, there was a close association between IL6 mRNA expression and the differentiation of mesenchymal cells into osteoblasts. TGF beta 1 expression was localized to osteoblasts apposed to bone or cartilage matrix; the intensity of expression correlated with matrix secretion. Chondroblasts and chondrocytes expressed lower but significant levels of TGF beta 1 mRNA; the expression was lost with the progression to calcifying cartilage. The three cytokines studied were differentially expressed both temporally and spatially, suggesting different roles for each in osteoblast and chondrocyte function.
Subcellular distribution of adenosine deaminase and adenosine deaminase-complexing protein in rabbit kidney: implications for adenosine metabolism.
Authors: Schrader W P , West C A , Rudofsky U H , Samsonoff W A
Published: April 2011

We evaluated the age-related distribution of adenosine deaminase (ADA) and adenosine deaminase-complexing protein (CP) in rabbit kidney by immunohistochemical staining procedures. Paraffin- or resin-embedded tissue from rabbits < 1 week-4 years of age were stained by the peroxidase-anti-peroxidase (PAP) method for ADA and CP. With the exception of neonates, the qualitative staining pattern of each protein remained generally constant with age. In the cortex, distal tubules, blood vessels, histiocytes, and epithelial cells lining Bowman's capsule stained for ADA. Proximal tubules and glomeruli were positive for CP. In contrast to the segregated pattern in the cortex, staining for ADA and CP overlapped in the corticomedullary junction. ADA and CP co-localized on the brush border of tubule cells of the S3 segment. In the cytoplasm of these cells, staining for ADA was characterized by scattered punctuate deposits of peroxidase reaction product. In some instances these punctuate deposits also appeared to be positive for CP. In medulla, epithelial cells of the thin limb were positive for both ADA and CP, whereas papillary collecting ducts stained only for CP. These results document the age-related, tissue-specific expression and localization of ADA in renal tissue, features that probably reflect the crucial role played by the enzyme in adenosine/deoxyadenosine catabolism. In addition, colocalization of ADA and CP on the brush border of cells in the S3 segment of proximal tubules provides support for the hypothesis that one function of CP may be to position ADA on the plasma membrane of specific cell populations, further expanding the enzyme's utility in nucleoside metabolism.
Androgen receptor immunostaining and its tissue distribution in formalin-fixed, paraffin-embedded sections after microwave treatment.
Authors: Iwamura M , Abrahamsson P A , Benning C M , Cockett A T , di Sant'Agnese P A
Published: April 2011

We used microwave (MW) oven heat treatment to unmask human androgen receptor (AR) immunostaining in formalin-fixed, paraffin-embedded tissue. Prostate tissue was used as an AR-positive control. Tissue sections were boiled in citrate buffer in a conventional MW oven for 30 min, followed by immunostaining with a validated murine monoclonal antibody (MAb), F39.4.1, raised against a peptide included in the N-terminal domain of the 100 KD human AR. AR immunostaining was localized to the nuclei of prostate secretory epithelial cells but was weak or absent in basal cells and of variable intensity in the stromal cells. Slides exposed to less than 10 min of MW heat treatment or none at all manifested no AR immunoreactivity. Tissue morphology was well preserved. Immunohistochemical determination of AR status in a wide variety of human tissues was consistent with that previously reported by others using frozen sections. MW heat treatment of tissue samples in an excellent method of localizing AR antigenicity, enabling immunohistochemical evaluation of AR status in formalin-fixed, paraffin-embedded material.
Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.
Authors: Gao K , Giffin B F , Morris R E , Cardell E L , Cardell R R
Published: April 2011

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.
Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA.
Authors: Sasaki K , Kurose A , Ishida Y , Matsuta M
Published: April 2011

We describe a method for measuring the size of the S-phase fraction in human tumor tissue sections using an antibody to PCNA (PC10). Although treatment with Triton X-100 before fixation extracted a large amount of PCNA from the cells even in frozen tissue sections, PCNA remained exclusively in S-phase cells. Immunohistochemical staining of PCNA after the detergent treatment allowed estimation of the S-phase fraction in solid tumors. The validity of the method was directly proven by double staining of bromodeoxyuridine (BrdU) and PCNA in HeLa cells. The PCNA-positive cells were identical with BrdUrd-positive cells after the detergent treatment. In contrast, almost all HeLa cells in the exponentially growing phase were positive for PC10 without treatment with Triton X-100.
Distribution of immunoreactive alpha- and beta-subunit isoforms of Na,K-ATPase in the gerbil inner ear.
Authors: McGuirt J P , Schulte B A
Published: April 2011

Biochemical and histochemical studies have demonstrated abundant Na,K-ATPase in the inner ear and provided new information concerning the ion transport capacities of specialized cell types. To extend these earlier observations, we immunostained inner ears from adult gerbils with antibodies specific for the three known alpha- and the two known beta-isoforms of Na,K-ATPase. Different inner ear cell types contained specific and distinct combinations of alpha- and beta-subunit isoforms. Strial marginal cells and vestibular dark cells expressed the alpha 1- and beta 2-isoforms, whereas other positive epithelial cells expressed alpha 1 in combination with beta 1. Ganglion neurons and their peripheral processes showed positive immunostaining for the alpha 3- and beta 1-subunit isoforms. Subpopulations of fibrocytes in the spiral prominence, suprastrial and supralimbal regions, and vestibular system expressed either the alpha 1- or alpha 2-isoform, or both. The differential expression of Na,K-ATPase subunit isoforms presumably reflects different K+ and Na+ transport capacities among inner ear cell types which, working in concert, serve to generate and maintain the unique ionic and electrical environment in the mammalian inner ear.