bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of immunostained ACTH1-39 fibers and CRF neurons in the paraventricular nucleus of rat hypothalamus: application of avidin-glucose oxidase to dual immunostaining procedures.
Authors: Piekut D T
Published: April 2011

Dual staining immunocytochemical procedures were used to elucidate the distribution and potential anatomic relationship of corticotropin releasing factor (CRF)-containing cell bodies and central opiocortin fibers in the paraventricular nucleus (PVN) of rat hypothalamus. Double-immunostained preparations employed antibodies and the peroxidase-antiperoxidase (PAP) technique as a first sequence in the immunostaining protocol, followed by antibodies and glucose oxidase-conjugated avidin as the second sequence, yielding rich brown and vibrant blue reaction products respectively. Distinctive features of this new dual immunostaining technique and its applicability to the study of co-localization and coexistence of neuropeptides are discussed. This study demonstrates immunostained ACTH1-39 fibers in intimate anatomic proximity to, and often surrounding in remarkable density, CRF-containing cells localized in distinct subnuclei of the PVN. It appears that the central opiocortin system selectively innervates CRF neurons in the PVN.
A cytochemical stain for glutathione in rat hepatocytes cultured on plastic.
Authors: Larrauri A , López P , Gómez-Lechón M J , Castell J V
Published: April 2011

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.
Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry.
Authors: Gonatas N K , Gonatas J O , Stieber A , Ternynck T , Avrameas S
Published: April 2011

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.
Endogenous peroxidase activity in human cutaneous and adenoidal mast cells.
Authors: Escribano L M , Gabriel L C , Villa E , Navarro J L
Published: April 2011

We have studied peroxidase activity in human cutaneous and adenoidal mast cells using different methods, in order to determine the optimal technical conditions for its demonstration. In 1.25% glutaraldehyde-fixed cells, no peroxidase activity was seen. On the contrary, in tannic acid-aldehyde-fixed cells or in unfixed cells peroxidase activity was revealed independently of the DAB concentration or the incubation time in DAB medium. The reaction product was localized in perinuclear cisternae and endoplasmic reticulum. Granules were always unreactive with all techniques employed. Golgi apparatus was generally negative and only occasional cells exhibited one or two positive peripheral cisternae. This activity appears sensitive to fixation by glutaraldehyde and is inhibited by 3-amino-1,2,4-triazole (AMT) and by lack of H2O2 or DAB in the incubation medium, but not by potassium cyanide, sodium azide, or sodium pyruvate, at the concentrations used. The peroxidase activity described in this report is an endogenous peroxidase and is not related to uptake of exogenous peroxidase by mast cells. It can therefore be considered as an ultracytochemical marker of human mast cells.
Improved fixation and cobalt-glucose oxidase-diaminobenzidine intensification for immunohistochemical demonstration of corticotropin-releasing factor in rat brain.
Authors: Sakanaka M , Shibasaki T , Lederis K
Published: April 2011

An optimal fixation method and intensification procedure may be required in brain immunohistochemistry to obtain intense and widespread staining for a specific antigen, in cases where ordinary fixation and conventional immunohistochemistry result in only partial demonstration of the antigen. In the present study of localization of corticotropin-releasing factor immunoreactivity (CRFI) in rat brain, the importance of such intensification is shown. We describe a fixation procedure in which perfusion of rat brain with Bouin's solution is followed by a PBS wash and a further perfusion with either Zamboni's fluid or 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), for subsequent investigation of the detailed localization of CRFI in cerebral cortex and subcortical structures. The cobalt-glucose oxidase-diaminobenzidine (Co-GOD) intensification method has been modified to increase the sensitivity of immunostaining by reducing the concentration of glucose oxidase, which is added to the final incubation solution as a generator of hydrogen peroxide. The use of cobalt acetate instead of cobalt chloride appears to slightly suppress background staining in the Co-GOD method. Combination of the two modified procedures was applied to visualize intense and widespread CRFI in a variety of rat brain regions, including median eminence, cerebral cortex, and central amygdaloid nucleus.
Intracellular localization of thymosin alpha 1 by immunoelectron microscopy using a monoclonal antibody.
Authors: Auger C , Stahli C , Fabien N , Monier J C
Published: April 2011

Distribution of thymosin alpha 1 in normal mice (OF1) or autoimmune mice (NZB) was investigated using immunocytochemical techniques on sections of GMA- and Epon-embedded mouse thymuses. A monoclonal antibody directed against synthetic thymosin alpha 1 was used. With the immunofluorescence assay, patchy staining of thymosin alpha 1 was found in the cytoplasm of epithelial cells of the subcapsullary and medullary zones of OF1 thymus. In NZB thymus, the fluorescent pattern was less precisely localized. At the electron microscopic level, immunolabeling of Epon-embedded ultra-thin sections revealed ferritin in some vacuoles of epithelial cells. Ferritin labeling in OF1 thymus was found in several small vacuoles of the same cell, but was present in large, dense vacuoles in NZB thymus. These differences might reflect differences in the secretory process of thymic hormone.
Preparation of colloidal gold-labeled agarose-gelatin microspherules for electron microscopic studies of phagocytosis in cultured cells.
Authors: Gao K X , Huang L
Published: April 2011

Agarose-gelatin microspherules about 0.5 micron or larger are prepared with emulsification of 4% agarose-gelatin sol containing 0.2 M N-octylglucoside in an organic phase composed of cyclohexane, egg lecithin, Span 80, and ethanol, followed by extraction of lipophilic components with cyclohexane and ether. Colloidal gold particles are then introduced into microspherules using gold chloride reacting at room temperature with tannic acid in a specified concentration range. After they have been coated with bovine serum albumin or mouse IgG, colloidal gold-labeled microspherules can be readily phagocytized by mouse L-cells and P388 cells after incubation for several hours. In addition to their use as a novel marker for phagocytosis, we discuss other potential uses for these colloidal gold-labeled microspherules.
Microspectrofluorometric comparison of benzo(a)pyrene and dibenzo(c,h)acridine metabolism in single living 3T3 fibroblasts.
Authors: Lahmy S , Salmon J M , Viallet P
Published: April 2011

The purpose of this study was to determine if dibenzo(c,h)acridine could be used as a probe for the N-heterocyclic aromatic compound detoxification system. The determination was achieved by cell population studies (histograms) using microspectrofluorimetry on single living cells. The results show a similar metabolic distribution pattern between dibenzo(c,h)acridine and benzo(a)pyrene, whereas important differences appear between 6-aminochrysene and dibenzo(c,h)acridine, respectively amine aromatic and N-heterocyclic aromatic compounds. No differences were observed in the metabolism of benzo(a)pyrene in untreated cells and those treated with constant concentrations of dibenzo(c,h)acridine. However, inhibition of dibenzo(c,h)acridine metabolism was observed in the presence of benzo(a)pyrene, indicating that this compound should be a preferential substrate for a cell detoxification system.
Rapid preparation of fresh-frozen undecalcified bone for histological and histochemical analysis.
Authors: Aaron J E , Carter D H
Published: April 2011

An increasing need to be able to relate hard tissue morphology to its histochemistry, and the lack of an adequate methodology, prompted this investigation of polyvinylpyrrolidones of varying chain length as supporting films for the cryomicrotomy of fresh undecalcified cortical and trabecular bone. We developed a method that parallels histological procedures but is biochemically inert and functions at -25 degrees C. Using an LKB heavy-duty cryomicrotome, large numbers of serial sections suitable for histochemistry, immunocytochemistry, or rapid histology can be prepared with ease. Sections from mouse skull, ox vertebra, and human iliac crest illustrate the versatility of the method, and have been examined for the preservation of general morphology and of histochemical activity of those enzymes most prominent in remodeling. The procedure does not preclude the subsequent application of standard hard-tissue embedding methods to the remaining material. Comparison with plastic-embedded bone shows not only close similarities in the structural preservation of the frozen tissue, making possible histomorphometry, but also shows some interesting staining differences. These include the absence of differentiation of the calcification front with toluidine blue stain in fresh frozen specimens, and the presence in frozen osteoid tissue of specific methylene blue metachromasia missing from plastic preparations.
A modified periodic acid-thiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by transmission electron microscopy.
Authors: Lo H K , Malinin T I , Malinin G I
Published: April 2011

Oxidation of araldite-embedded liver sections by 1% w/v aqueous H5IO6 for 15 min and a 5-min reaction of carbonyls with 1% w/v thiocarbohydrazide in 10% v/v acetic acid was employed for subsequent staining of glycogen with silver-proteinate (S-P). The network of branching intracellular glycogen aggregates was revealed by 15-min staining with S-P, whereas 24 hr incubation in S-P was necessary to enhance the contrast of glycogen inclusions. We conclude that the proposed modification of glycogen staining readily affords the means for its localization at a desired level of contrast and resolution.
Cellular and subcellular distribution of laminin in adult rat anterior pituitary.
Authors: Vila-Porcile E , Picart R , Tixier-Vidal A , Tougard C
Published: April 2011

Following the immunodetection of laminin in basal laminae and within some glandular cells of adult rat pituitary (Tougard et al., 1985: In Vitro 21:57), the present work had a double purpose: (a) adjustment of optimal conditions to detect laminin at both the cellular and subcellular level with an immunoperoxidase pre-embedding procedure, and (b) identification of the hormonal specificity of the laminin-containing cells, based on development of a "two-step" method combining the pre- and post-embedding approaches. In addition to extracellular localizations (basal laminae and connective tissue compartment), laminin was detected intracellularly within membrane-bounded compartments in all endocrine cell types and in the folliculo-stellate cells. It was found in rough endoplasmic reticulum cisternae, in a few Golgi saccules, and in vesicles or vacuoles. Labeled secretory granules were observed in gonadotrope, thyrotrope, and corticotrope cells but were very scarce in prolactin cells and absent in somatotrope cells. In addition, exocytotic profiles of labeled granules were frequent, suggesting a granular pathway for laminin export. Labeled vesicles, heterogeneous in size and shape, were observed mostly in prolactin cells. They might represent either a vesicular pathway for laminin export or an endocytic route for membrane-bounded laminin. These results bring complementary data to the concept that epithelial cells elaborate their basement membranes (Pierce and Nakane, 1967: Lab Invest 17:499). Moreover, the subcellular distribution of laminin within organelles involved in the biosynthesis, transport, and even in the storage of secretory products suggests that laminin might be exported by glandular pituitary cells according to different pathways.
Ligand-dependent redistribution of the IgA receptor on cultured rat hepatocytes and its disturbance by cytochalasin B.
Authors: Gebhardt R , Robenek H
Published: April 2011

The topography and dynamics of IgA-secretory component (SC) complexes on the surface of cultured hepatocytes and its disturbance by cytochalasin B were investigated using the colloidal gold technique in conjunction with surface replication. The distribution of IgA-gold conjugates after incubation at 4 degrees C was similar in normal and cytochalasin B-treated hepatocytes and was characterized by diffusely scattered single and clustered particles, the latter often associated with coated pits. After raising the temperature to 37 degrees C, redistribution of particles and their gradual uptake into coated vesicles was observed in control cultures. This ligand-induced redistribution led to a progressive gathering of single and grouped particles in larger clusters (50-200 particles), which appeared to be the site of the most intensive endocytotic activity. In contrast, huge patches of IgA-gold conjugates were formed at the cell periphery of cytochalasin B-treated hepatocytes within 20-60 min at 37 degrees C, while central areas were cleared. Patch formation was triggered by binding of both unlabeled and labeled IgA, but could not be observed with the unoccupied receptor as demonstrated by gold-labeled antibodies against SC. These results show that the topography of SC is markedly changed by binding of its ligand, IgA, and suggest that the dynamics of the IgA-SC complexes in hepatocyte plasma membrane are affected by microfilaments.
Transition from mitosis to interphase in sea urchin first division: immunofluorescence studies of tubulin distribution in methacrylate sections.
Authors: Harris P J , Rubin B P
Published: April 2011

Previous immunofluorescence studies of microtubule distribution in fertilized sea urchin eggs have suffered from poor resolution caused by cell thickness, unavoidable artifacts resulting from excessive flattening, or extraction by detergents of membranes and other lipid-containing structures that may be of interest in relation to the microtubules. To avoid these difficulties, we have developed a fixation and embedding protocol based on buffered paraformaldehyde fixation and butyl-methyl methacrylate embedment, which allows immunofluorescence staining of 0.5-1 micron sections. Polymerization artifacts are reduced by polymerizing the methacrylate at a relatively low temperature (40-45 degrees C) and by flat embedding for more uniform polymerization. Using this method, we have examined mitotic stages in the first cleavage cycle of the sea urchin Strongylocentrotus purpuratus. We provide evidence that the interphase microtubules that appear after first division are not derived from the mitotic asters but are new structures growing from organizing centers within the degenerating mitotic asters. During the transition from mitosis to interphase, there is a temporary overlap of old and new microtubules to form a very large composite aster at telophase before the old structure finally disappears.
Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.
Authors: Posthuma G , Slot J W , Geuze H J
Published: April 2011

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.
Labeling of structural elements at the ventral plasma membrane of fibroblasts with the immunogold technique.
Authors: Nicol A , Nermut M V , Doeinck A , Robenek H , Wiegand C , Jockusch B M
Published: April 2011

We investigated the organization of two structural elements (microfilaments and clathrin coated structures) at the ventral plasma membrane in fibroblasts. The methods employed included exposure of the inner face of the ventral plasma membrane by lysis-squirting, labeling the exposed structures with specific antibodies conjugated with colloidal gold, and platinum-carbon replica techniques. The results demonstrate that this regimen combines good ultrastructural preservation with unambiguous identification of structural elements associated with the plasma membrane. This method is therefore reliable and can be generally used to explore in detail the ultrastructural organization of the plasma membrane.
Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation.
Authors: Salama A H , Zaki A E , Eisenmann D R
Published: April 2011

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.
Epithelial membrane polarity: a stable, differentiated feature of an established human breast carcinoma cell line MCF-7.
Authors: van Deurs B , Zou Z Z , Briand P , Balslev Y , Petersen O W
Published: April 2011

Carcinoma cells typically show little or no polarity as compared to normal, differentiated epithelial cells. We have studied polarity in two established human breast carcinoma cell lines, T47D and MCF-7, by various techniques (electron microscopic enzyme- and immunocytochemistry, freeze-fracture) and show that one of them (MCF-7) is characterized by a high degree of polarity. Thus, in contrast to T47D cells, MCF-7 cells in monolayer culture form apical tight junctions, do not allow a ricin-horseradish peroxidase conjugate, which binds to terminal galactose residues on the apical surface, to stain the basolateral membrane domain, and express a surface antigen (MFGM-A) only in the apical surface membrane domain, as do normal mammary epithelial cells in vivo. This polarization is independent of a basement membrane, since it is maintained when MCF-7 cells, which do not deposit type IV collagen themselves, are grown directly on plastic. Moreover, even though MCF-7 cells express estrogen receptors rather homogeneously, estrogen has no effect on this polarity, neither in vitro nor after transplantation to nude mice. We conclude that polarity is a stable, differentiated feature of MCF-7 cells.
Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules.
Authors: Frederickson C J , Pérez-Clausell J , Danscher G
Published: April 2011

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.
Distribution of basal lysosomes in exocrine acinar cells.
Authors: Oliver C , Yuasa Y
Published: April 2011

We examined the distribution of trimetaphosphatase (TMPase)-positive basal lysosomes in pancreas, parotid, submandibular, sublingual, and exorbital lacrimal glands from rats, rabbits, and guinea pigs. The location of the basal lysosomes was compared to that of the acid phosphatase (AcPase)-positive lysosomes. In all of the tissues examined from rat and rabbit, AcPase activity was localized primarily to the Golgi region. Reaction product was localized in GERL, immature secretory granules, and lysosomes lying adjacent to the Golgi apparatus. TMPase activity was found in basal lysosomes and in occasional elongated lysosomes adjacent to the Golgi apparatus. In guinea pig, the distribution of TMPase activity was identical to that seen in the other two species, but a significant number of lysosomes in the basal region of the cells also contained AcPase activity. These results confirm and extend our previous finding (J Histochem Cytochem 31:1209, 1983) that exocrine acinar cells possess two distinct populations of lysosomes. The lysosomes in the Golgi region contain both AcPase and TMPase activity, whereas those in the basal portion of the cells are reactive predominantly for TMPase. The functional significance of the two populations of lysosomes is not understood at present.
Ultrastructural localization of arylsulfatase C activity in rat kidney.
Authors: Kawano J , Aikawa E
Published: April 2011

Metal precipitation techniques for ultrastructural demonstration of arylsulfatase C activity were studied in rat kidney. Possible substrates for the techniques were biochemically tested with regard to their velocity of enzymatic hydrolysis and their specificity for arylsulfatase C. Effects of buffers and capturing metals were also examined. The results of these biochemical studies were then verified histochemically. Incubation in a medium containing 1 mM 4-methylumbelliferyl sulfate, 1% barium chloride, 0.1 M imidazole-HCl buffer (pH 7.5), and 5% sucrose achieved identifiable results in adequately fixed kidney. Precipitation of barium sulfate was localized mainly in the endoplasmic reticulum and perinuclear cisterns of the epithelial cells in the descending portions of proximal tubules.
Whole-body autoradiographic distribution of exogenously administered renal renin in rats.
Authors: Kim S , Iwao H , Nakamura N , Ikemoto F , Yamamoto K
Published: April 2011

We studied, by whole-body autoradiography, the distribution of exogenously administered renal renin in rat. Rat renal renin was completely purified and labeled with 125I ([125I]-renin) and was then injected into the tail veins of conscious rats at a dose of 30 microCi, 430 ng. After various intervals, rats were killed by an overdose of ether, the whole body rapidly frozen in acetone-dry ice, and autoradiography performed on sagittal whole-body sections. To remove breakdown products ([125I]-tyrosine and free 125I) from [125I]-renin, sections were treated with perchloric acid solution. The main accumulation of [125I]-renin acid-insoluble radioactivity was observed in liver and renal cortex. The accumulation in these organs was already evident 2 min after the injection, reached a maximum level by 15 min, then gradually decreased. A small amount of [125I]-renin was also evident in spleen, bone marrow, and adrenal gland. Thirty min after the injection, radioactivity began to appear in the thyroid gland, stomach, and small intestine, but disappeared with acid treatment, except in the thyroid. Radioactivity was negligible in other organs including brain, submaxillary gland, lung, heart, and testis. These autoradiographs clearly demonstrate that exogenously administered renal renin is distributed mainly in the liver and renal cortex.
Immunohistochemical detection of proliferating cells in vivo.
Authors: deFazio A , Leary J A , Hedley D W , Tattersall M H
Published: April 2011

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.
Immunohistochemical localization of acetylcholine receptors at human endplates using a monoclonal antibody.
Authors: Smit L M , Veldman H , Jennekens F G
Published: April 2011

We describe a simple indirect immunohistochemical method for localization of acetylcholine receptors (AChR) in motor endplates at the light and electron microscopic level. This method involves the use of a monoclonal antibody directed against the main immunogenic region (MIR) of AChRs and is applicable to periodate-lysine-paraformaldehyde (PLP)-fixed tissue. We discuss the advantages of this method, as compared with the alpha-bungarotoxin-immunoperoxidase technique, and stress its value for diagnostic investigations of motor point biopsies from patients with neuromuscular transmission disorders.
Increased ATPase acid stability in type 1 fibers of rat soleus.
Authors: Smith A , Carter N , Jeffery S
Published: April 2011

Type 1 fibers in skeletal muscle have been considered homogeneous on the basis of their acid-resistant ATPase activity. We have found that the Type 1 fibers in rat soleus are more resistant to prolonged acid pre-incubation than are those in other muscles.
Thyroidectomy significantly alters carbonic anhydrase III concentration and fiber distribution in rat muscle.
Authors: Jeffery S , Carter N D , Smith A
Published: April 2011

Thyroidectomy has a dramatic effect on rat muscle, greatly increasing the number of Type I fibers and the concentrations of carbonic anhydrase III (CAIII) in the muscle. Carbonic anhydrase III is not confined to the Type I fibers, as was previously believed, but also occurs in fibers that exhibit a level of ATPase staining less than that of 2A fibers but greater than 2B. These fibers are rare in normal muscle but become numerous after thyroidectomy, when they stain heavily for CAIII.
Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells.
Authors: Tanaka K , Akayama M , Yamamoto A , Omori K , Tashiro Y
Published: April 2011

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.
Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers.
Authors: Ross C D , Godfrey D A
Published: April 2011

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.
Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue.
Authors: Jorgensen A O , McGuffee L J
Published: April 2011

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.
Fixation and immunofluorescent analysis of creatine kinase isozymes in embryonic skeletal muscle.
Authors: Robinson M M
Published: April 2011

Pectoral muscles from chicken embryos of various ages were examined with immunofluorescent and radiolabeled probes for the presence of brain-type creatine kinase (B-CK), muscle-specific creatine kinase (M-CK), muscle-specific myosin heavy chain (MHC), and cycling cells. The diffusible creatine kinase isozymes were not detectable by indirect immunofluorescence after standard histological fixation of embryonic muscle. However, a fixation procedure was devised that permitted immunodetection of the creatine kinase isozymes (particularly B-CK) in embryonic tissue from all stages of development studied. B-CK, M-CK, and MHC were all detected in post-mitotic muscle cells, but only B-CK was detected in cycling cells. Correlations between these findings and in vitro observations of a deterministic muscle lineage are discussed.
Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones.
Authors: Mark M P , Prince C W , Oosawa T , Gay S , Bronckers A L , Butler W T
Published: April 2011

Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.
Immunocytochemical detection of anti-müllerian hormone in Sertoli cells of various mammalian species including human.
Authors: Tran D , Picard J Y , Campargue J , Josso N
Published: April 2011

An immunocytochemical method, based on the use of a polyclonal antibody raised against purified bovine anti-Müllerian hormone (AMH), was used to detect AMH in Sertoli cell cytoplasm of various mammalian species, including human. Immunopurification of antiserum by AMH-affinity chromatography, although not mandatory, leads to better results and increased sensitivity. In human testicular tissue, AMH is detectable up to 6 years of age. In rats, AMH production is initiated at 13 days post coitum, peaks between 15 and 17 days, and is no longer detectable 1 week after birth. The reaction is strongest in Sertoli cells of calves, sheep, goats, and pigs, species characterized by a high degree of development of the rough endoplasmic reticulum. It is fainter in human, rat, rabbit, and cat Sertoli cells, in which the rough endoplasmic reticulum is not as abundant. This correlation is not unexpected, in view of the localization of reaction product in this cytoplasmic organelle. Preliminary results indicate that there may be a relationship between the amount of immunoreactive AMH present in testicular biopsies of intersex patients and the degree of regression of the Müllerian duct on the ipsilateral side. This may help to elucidate whether persistence of Müllerian ducts results from lack of testicular production of AMH or from peripheral resistance of the Müllerian primordia to the hormone.
Autoradiographic demonstration of in vivo sialylation of endogenous acceptors at the microvillar surface of intestinal columnar cells after intraluminal administration of CMP-[3H]-sialic acid.
Authors: Bennett G , Haddad A , Leblond C P
Published: April 2011

To investigate the presence of glycosyltransferase activity at the apical surfaces of columnar cells in small intestine, CMP-[3H]-sialic acid was injected into the lumen of a ligated segment of rat jejunum; 5 min later the tissue was fixed and processed for light microscopic autoradiography. After a 3-6-month exposure, an autoradiographic reaction appeared over the microvillar surfaces of columnar cells, indicating the presence of surface sialyltransferase activity accompanied by endogenous acceptors. When CMP-[3H]-sialic acid was injected into the posterior chamber of rat eye or the lumen of mouse gallbladder, no autoradiographic reaction was observed at the surfaces of the cells facing these cavities. After injection of UDP-[3H]-galactose into the same three sites, an autoradiographic reaction was observed in the Golgi regions of the various epithelial cells, but not along their apical surfaces. Competition experiments using unlabeled galactose indicated that [3H]-galactose had been released from the nucleotide and had entered the cells to be incorporated into the Golgi apparatus.
Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse.
Authors: Cammer W , Tansey F A
Published: April 2011

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.
Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells.
Authors: Pakkanen R , Hedman K , Turunen O , Wahlström T , Vaheri A
Published: April 2011

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.
Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement.
Authors: Bronckers A L , Gay S , Finkelman R D , Butler W T
Published: April 2011

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.