bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical localization of cellCAM 105 in rat tissues: appearance in epithelia, platelets, and granulocytes.
Authors: Odin P , Asplund M , Busch C , Obrink B
Published: April 2011

CellCAM 105 is an integral membrane glycoprotein, with apparent Mr 105,000, which has been purified from rat liver plasma membranes. It consists of two structurally similar, highly glycosylated polypeptide chains and is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In this communication we report on the distribution and cell surface location of cellCAM 105 in rat tissues, obtained by using highly sensitive immunodetection systems based on complex formation between biotinylated antibodies, biotinylated peroxidase and avidin, or on antibodies coupled to alkaline phosphatase. CellCAM was found in many organs and organ systems, including liver, kidney, blood, blood vessels, glands, respiratory system, and gastrointestinal tract. It was mainly localized to epithelial structures but showed a varying cell surface distribution. In some cell types it was predominantly localized to cell-cell contact areas. In other cell types the highest concentrations were seen in brush-border areas containing densely packed microvilli. In addition to epithelial structures, cellCAM 105 was found in rat platelets, where it became strongly expressed on the cell surfaces after activation with ADP or collagen, suggesting that it might be involved in platelet adhesion and/or aggregation mechanisms. Granulocytes also contained cellCAM 105. By SDS-PAGE/immunoblotting, significant differences were found in the apparent Mr values of cellCAM 105 in different tissues. The collected data suggest that cellCAM 105 participates in several different cell surface membrane interactions, of which the common denominator might be membrane-membrane binding.
Antibodies to mouse lung capillary endothelium.
Authors: Rorvik M C , Allison D P , Hotchkiss J A , Witschi H P , Kennel S J
Published: April 2011

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.
Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas.
Authors: Watanabe M , Watanabe T , Ishii Y , Matsuba H , Kimura S , Fujita T , Kominami E , Katunuma N , Uchiyama Y
Published: April 2011

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.
Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue.
Authors: Murray G I , Burke M D , Ewen S W
Published: April 2011

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.
Gold probe choice in simultaneous detection of human lymphocyte surface antigens at the ultrastructural level.
Authors: Hoogeveen Y L , Smit J W , Blom N R , Halie R M
Published: April 2011

A double immunogold-labeling method in immunoelectron microscopy was used for simultaneous detection of two antigens by monoclonal antibodies [OKT 8 (CD 8), anti-Leu-7, anti-Leu-11b (CD 16)] on lymphocytes in suspension. The combination of gold probe size (5 nm and 15 nm) and monoclonal antibody was found to be decisive for detecting double-labeled cells with the OKT 8+, Leu-11b+ phenotype. The combinations of OKT 8 labeled with the 5-nm gold probe (OKT 8(5] and anti-Leu-11b with the 15-nm gold probe (Leu-11b15) gave double-labeled cells; the reverse situation, using OKT 8 with a 15-nm gold probe (OKT 8(15] and anti-Leu-11b with a 5-nm gold probe (Leu-11b5), did not. Double-labeled OKT 8+, Leu-7+ cells were detected irrespective of which gold probe combination was applied. Our findings indicate that although the double immunogold-labeling method is well suited for study of lymphocyte subsets, it is important to determine suitable combinations of gold probe sizes and monoclonal antibodies for the lymphocyte subset under study, taking into account surface antigen density, so that double labeling ensues.
Hematoporphyrin/DAPI staining: simplified simultaneous one-step staining of DNA and cell protein and trial application in automated cytological screening by flow cytometry.
Authors: Takahama M , Kagaya A
Published: April 2011

We describe a procedure for simplified, simultaneous one-step staining in 10 min for DNA and cell and tissue proteins using a newly developed staining solution containing 0.03% hematoporphyrin (HP) with 0.001% DAPI [or with Hoeschst 33342 (HO)]. These HP/DAPI or HP/HO solutions were especially developed to facilitate a trial of automated cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of DNA and that of protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of DNA and protein. On flow cytometry, a single UV beam (370 nm) from the argon laser was used for excitation of both dyes. Measurement of DNA was done using a 470-nm bandpass filter and of protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of protein and DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and cancer cells easier.
Immunocytochemical localization of cytosol 5'-nucleotidase in chicken liver.
Authors: Yokota S , Oka J , Ozasa H , Itoh R
Published: April 2011

We investigated the localization of cytosol 5'-nucleotidase in chicken liver by use of a pre-embedding immunoenzyme technique. Cytosol 5'-nucleotidase was purified from chicken liver and a monospecific antibody to this enzyme was raised in a rabbit. Fab fragments of the antibody were conjugated with horseradish peroxidase. Tissue sections of the fixed chicken liver were incubated with the peroxidase-Fab fragments, followed by DAB reaction for peroxidase. By light microscopy, dark-brown staining was present in the cytoplasm of parenchymal cells, Kupffer cells, and endothelial cells. The latter two types of cells were stained more strongly than the former. By electron microscopy, reaction deposits were present in the cytoplasmic matrix but not in cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes, or in nuclei. In control sections incubated with peroxidase-conjugated Fab fragments from non-immunized rabbit, no specific reaction was noted. The results indicate that cytosol 5'-nucleotidase is contained more in the sinus-lining cells and less in the parenchymal cells, and that the enzyme is present in the cytoplasmic matrix of these cells.
Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.
Authors: Akagi S , Yamamoto A , Yoshimori T , Masaki R , Ogawa R , Tashiro Y
Published: April 2011

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.
Multiple-reaction cycling: a method for enhancement of the immunochemical signal of monoclonal antibodies.
Authors: Linsenmayer T F , Fitch J M , Schmid T M
Published: April 2011

Most current studies using immunochemical and immunohistochemical procedures to detect antigen-antibody complexes employ some type of indirect method. Such procedures afford signal amplification because several marker-conjugate molecules can bind to each primary antibody molecule. We have observed that for monoclonal antibodies an even greater amplification can be afforded simply by performing two (or more) reaction cycles (i.e., primary antibody, secondary antibody-primary antibody, secondary antibody-etc). In the present report, we demonstrate the utility of this method for immunohistochemical (immunofluorescence) and immunochemical (ELISA: enzyme-linked immunosorbent assay) procedures employing well-characterized monoclonal antibodies directed against avian type IV (basement membrane) collagen.
High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.
Authors: Londoño I , Bendayan M
Published: April 2011

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.
Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits.
Authors: Weis K G , Polito V S , Labavitch J M
Published: April 2011

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.
Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques.
Authors: Nagy-Oltvai Z , Brady T , Hsiung G D
Published: April 2011

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.
Connective tissue of rat lung. II: Ultrastructural localization of collagen types III, IV, and VI.
Authors: Amenta P S , Gil J , Martinez-Hernandez A
Published: April 2011

We localized collagen types III, IV, and VI in normal rat lung by light and electron immunohistochemistry. Type IV collagen was present in every basement membrane examined and was absent from all other structures. Although types III and VI had a similar distribution, being present in the interstitium of major airways, blood vessels, and alveolar septa, as in other organs, they had different morphologies. Type III collagen formed beaded fibers, 15-20 nm in diameter, whereas type VI collagen formed fine filaments, 5-10 nm in diameter. Both collagen types were found exclusively in the interstitium, often associated with thick (30-35 nm) cross-banded type I collagen fibers. Occasionally, type III fibers and type VI filaments could be found bridging from the interstitium to the adventitial aspect of some basement membranes. Furthermore, the association of collagen type VI with types I and III and basement membranes suggests that type VI may contribute to integration of the various components of the pulmonary extracellular matrix into a functional unit.
Chick osteoblasts contain fluoride-sensitive acid phosphatase activity.
Authors: Lundy M W , Lau K H , Blair H C , Baylink D J
Published: April 2011

We used histological and biochemical methods to determine the cellular origin of bone matrix fluoride-sensitive acid phosphatase in chicken bone. Embryonic chicken calvariae were embedded in plastic and sections stained for acid phosphatase at various concentrations of substrate and fluoride. Acid phosphatase activity was observed in osteoblasts and osteoclasts but not in fibroblasts. Striking inhibition of osteoblastic acid phosphatase occurred at 100 microM fluoride, a concentration that had no apparent effect on osteoclastic acid phosphatase. Inhibition of osteoblastic and osteoclastic acid phosphatase by fluoride was also examined using extracts of embryonic chicken calvarial cells, mouse osteoblasts (MC3T3-El cell line), and purified chick osteoclasts, respectively. Fluoride is a partial competitive inhibitor of both chicken and mouse osteoblastic acid phosphatases, with apparent inhibition constants of 10-100 microM. These concentrations of fluoride correspond to those that increase bone formation in vitro and in vivo. In contrast, the apparent inhibition constant for fluoride of osteoclastic acid phosphatase was much higher (i.e., 0.5 mM). In summary, this study demonstrates that chicken osteoblasts contain an acid phosphatase that is sensitive to inhibition by low concentrations (i.e., microM) of fluoride.
Brain blotting: a method to detect multiple DNA copies in specific brain regions.
Authors: Hernandez Bronchud M , Webb S , Esiri M M
Published: April 2011

We developed a method to detect multiple DNA copies (both cellular and viral) in specific brain regions by blotting thick frozen sections onto nylon membranes. This was achieved by "printing" the frozen sections on standard blotting paper immediately after cryotome sectioning and performing blotting according to the standard Southern technique. A "replica" of the blotted section was obtained by keeping on the glass slide the next frozen section cut, which was then stained for conventional histopathological analysis and the cell nuclei counted to give an estimate of the total amount of DNA present in each section. The blotted membranes were then denatured and hybridized with a nick-translated Alu probe either at 42 degrees C with 50% formamide or at 68 degrees C without formamide. Brain sections from mice infected with Herpes simplex virus 1 (HSV1), blotted and hybridized with a nick-translated HSV1 probe, clearly showed the focal nature of the Herpes simplex infection, which was also demonstrated immunohistologically using a virus specific antiserum. This method of DNA detection, conveniently modified, might also be used to detect nuclear and cytoplasmic RNAs in specific coronal sections of whole brain before localization at high power by standard in situ techniques.
Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells.
Authors: Zou Z Z , Petersen O W , van Deurs B
Published: April 2011

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).
Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro.
Authors: Oliver C , Tolbert C L , Waters J F
Published: April 2011

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.
Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture.
Authors: Pavan A , Mancini P , Cirone M , Frati L , Torrisi M R , Pinto da Silva P
Published: April 2011

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.
Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin.
Authors: Hunter S J , Schraer H , Gay C V
Published: April 2011

We investigated the effects of calcitonin (CT) and parathyroid hormone (PTH) on the distribution of actin, tubulin, vimentin, and on cell size in cultured chick osteoclasts. In addition, we studied the effects of colchicine on intracellular acidity. Osteoclasts were isolated from the endosteum of 2-3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with CT for 30 min or PTH for 60 min and were observed after immunocytochemical staining of cytoskeletal proteins. In untreated cells, actin was found in both a filamentous and a punctate staining pattern, with indented or invaginated regions free of punctate spots. The tubulin distribution in untreated cells was characterized by a pattern of microtubules radiating from the cell center and running parallel to the cell edge. Vimentin staining was usually localized to the perinuclear area. There were no changes in cytoskeletal element distribution or morphology attributable to PTH treatment. Osteoclasts treated with CT were more irregularly shaped, contained more retraction fibers, and were more rounded, with a denser array of cytoskeletal elements in the cell center. In addition, the mean area of the CT-treated cells was significantly less than that of the untreated cells. The actin distribution after CT treatment was still characterized by both a filamentous and a punctate pattern. After CT treatment, vimentin staining appeared more centrally localized than in untreated cells and tubulin staining revealed microtubules which now extended to the retracted cell margin. These results indicate that isolated osteoclasts respond to CT by significant morphological changes which are reflected in the distribution of the major cytoskeletal elements. Disruption of the microtubular system by colchicine treatment also resulted in an initial increase in intracellular acidity, suggesting the involvement of microtubules in the movement of acid-laden vesicles to the exterior.
Acid giemsa technique for rapid identification of mitotic cells.
Authors: Dooley W C , Roberts J , Allison D C
Published: April 2011

We developed a rapid technique for differential staining of compacted chromatin as a tool for screening of large tissue culture cell populations for mitotic cells. With a combination of acid Giemsa staining and counterstaining, differential staining of mitotic cells and classification according to stage of mitosis can be accomplished at magnifications as low as x 50-100 (objectives of x 5-10). The mapped and classified cells can then be de-stained and re-studied for DNA content by Feulgen staining and/or for uptake of radioactive DNA precursors by autoradiography. The staining and de-staining procedures outlined do not affect the reproducibility and accuracy of DNA content measurements or measurements of radioactive uptake. Therefore, this technique can be used for cell kinetic analysis by the percentage labeled mitoses method and for cytophotometric studies of mitotic segregation.
Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin.
Authors: Fujimoto T , Ogawa K
Published: April 2011

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.
Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.
Authors: Milde P , Merke J , Ritz E , Haussler M R , Rauterberg E W
Published: April 2011

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.
Density gradient separation of two populations of lysosomes from rat parotid acinar cells.
Authors: Oliver C , Dromy R , Hart T K
Published: April 2011

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.
Ultrastructural localization of adenylate cyclase in chicken bone cells.
Authors: Yamamoto T , Gay C V
Published: April 2011

We investigated adenylate cyclase distribution in 6-day-and 3-week-old calvariae and in 6-day-old long bone metaphyses from chickens. Reaction product distribution was on the plasma membrane of osteoblasts, pre-osteoblasts, and forming osteocytes which contacted one another. Osteoclasts and mature osteocytes lacked reaction product. Six-day calvariae reacted less intensely than the other two tissues. In controls, reaction product was markedly diminished or eliminated by removal of forskolin or substrates, or by addition of the inhibitor 2',5'-dideoxyadenosine. The results indicate the importance of cyclic AMP in osteoblast regulation. Osteocytes and osteoclasts may involve alternate mechanisms as major regulatory systems.
Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.
Authors: Schrijvers A H , Frederik P M , Stuart M C , Burger K N , Heijnen V V , Van der Vusse G J , Reneman R S
Published: April 2011

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.
Delivery of lectin-labeled membrane to the trans-Golgi network and secretory granules in cultured atrial myocytes.
Authors: Iida H , Shibata Y
Published: April 2011

To examine whether and how internalized plasma membrane components are routed to the compartment of the biosynthetic-exocytic pathway in cultured atrial myocytes, the plasma membrane labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was traced electron microscopically by cytochemical detection of HRP. The WGA-HRP label was internalized via a coated pit-small vesicle pathway and reached vacuoles and endosomes by 3 min. Labeled endosomes comprised vacuoles and tubular elements containing reaction product. By 15 min, similar tubular structures containing reaction product accumulated in the area of the trans-Golgi network (TGN). The labeled TGN consisted of interconnected tubular elements, which often connected to atrial granules containing reaction product. In contrast, neither native HRP nor Lucifer Yellow reached Golgi elements or atrial granules. These results suggest that a proportion of the plasma membrane labeled with WGA-HRP is delivered to endosomes, from which tubules might bud off to transfer the tracer molecules to the TGN, where the lectin conjugate and associated membranes are packaged into atrial granules.
The known purified mammalian 2,4-dienoyl-CoA reductases are mitochondrial isoenzymes.
Authors: Hakkola E H , Autio-Harmainen H I , Sormunen R T , Hassinen I E , Hiltunen J K
Published: April 2011

The aim of this work was to determine the subcellular location of mammalian 2,4-dienoyl-CoA reductase, a key enzyme for degradation of polyunsaturated fatty acids by beta-oxidation. The enzyme was purified according to Kimura et al. (J Biochem 96:1463, 1984), and antibodies were raised in rabbits. Monospecific antibodies were obtained via purification on an affinity column. Immunoblotting of isolated rat liver mitochondria and peroxisomes with the monospecific reductase antibody showed that the antigen was located only in mitochondria. Immunocytochemical experiments with liver tissue, using the protein A-gold labeling technique, confirmed this result. The similarity of their characteristics suggests that the purified reductases described in the literature are the same isoenzyme. Consequently, since the rat enzyme was localized here to the mitochondria, purification and characterization of peroxisomal mammalian reductases remain to be achieved in the future. In addition, a significant induction also of mitochondrial reductase by clofibrate was observed in the immunoblotting experiments.
Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase.
Authors: Zoellner H F , Hunter N
Published: April 2011

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.
Immunohistochemical analysis of human neuronectin expression in normal, reactive, and neoplastic tissues.
Authors: Garin-Chesa P , Melamed M R , Rettig W J
Published: April 2011

Neuronectin (NEC1) is a human extracellular matrix protein of central nervous system (CNS) parenchyma found throughout the white matter of rostral brain segments (telencephalon, diencephalon, some areas of mesencephalon), but not in rostral CNS gray matter, most areas of mesencephalon, pons, cerebellum, medulla, spinal cord, or peripheral nerves. The present immunohistochemical study, using two monoclonal antibodies to distinct epitopes on the NEC1 molecule, examined NEC1 expression in normal non-neural tissues, malignant tumors of diverse histological types, non-malignant skin lesions, and dermal incision wounds. We show that (a) NEC1 is expressed in normal fetal precartilage blastemas and fetal and adult vascular and visceral smooth muscle, but not in most loose connective tissues and skeletal or cardiac muscle; (b) NEC1 is found along epithelial-mesenchymal junctions, with marked differences in prevalence and histological patterns in different organs; and (c) mesenchymal activation associated with wound healing, actinic keratosis, psoriasis, and neoplasia leads to strong induction of NEC1 expression. Parallel studies with cultured human cells suggest that region-specific NEC1 expression in normal developing tissues and localized induction in wound healing and disparate diseases is under the control of extrinsic signals provided by regulatory polypeptides.
Immunohistochemical localization of vimentin in the gerbil inner ear.
Authors: Schulte B A , Adams J C
Published: April 2011

Cells containing immunoreactive vimentin-type intermediate filaments (IF) were identified in paraffin sections and whole-mount preparations of the gerbil inner ear. Most connective tissue cells showed positive immunostaining, although one unusual class of stromal cell lacked vimentin. Several different types of epithelial cells contained high levels of vimentin. In the cochlea, Deiters' cells, inner phalangeal cells, Boettcher's cells, some outer sulcus cells, and the intermediate cells of the stria vascularis showed strong immunoreactivity. Strial basal cells exhibited weaker and less consistent staining. Neither inner nor outer hair cells were stained. In the vestibular system, hair cells with a morphology and location more characteristic of type I than of type II cells showed strong immunostaining for vimentin. Supporting cells in vestibular neurosensory epithelium stained with less intensity. These results were surprising because epithelial cells in vivo only rarely express vimentin-type IF. Although the functional significance of vimentin remains to be established, its presence in some but not other highly specialized cell types provides an excellent marker for investigating the lineage and morphogenesis of the complex inner ear tissues.
Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation.
Authors: Ichikawa M , Sasaki K , Ichikawa A
Published: April 2011

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.
Method for epitope selection of antisera for immunohistology.
Authors: Wedlich D
Published: April 2011

Polyclonal anti-laminin serum was affinity-purified on paraformaldehyde-fixed laminin on a nitrocellulose filter. The purified antibodies were tested for their specificity in immunohistological stainings on frozen sections of paraformaldehyde-fixed tissue. As compared to the initial polyclonal serum, the purified antibodies increased the specificity of antigen detection, since all background caused by nonspecific reactions was eliminated. This technique promises to be very useful for immunohistological analysis using light and electron microscopy.
Monoclonal antibodies against species-specific antigens in the chick central nervous system: putative application as transplantation markers in the chick-quail chimera.
Authors: Takagi S , Tsuji T , Kinutani M , Fujisawa H
Published: April 2011

With the recent progress in transplantation of neuronal tissues, cellular markers are needed to distinguish the grafted cells from the host. To generate monoclonal antibodies (MAb) recognizing species-specific antigens in the chick nervous system, we immunized mice with chick optic nerves and obtained 2 MAb which bind to chick but not to quail neural tissues. MAb-39B11 recognizes the cell surface antigen on the nerve fibers. MAb-37F5 recognizes the cytoplasmic components in several cell types, including ependymal cells and some large neurons. The utility of these MAb as markers for chick cells in the chick-quail chimeric brain and their advantages over conventional markers are discussed.
Distribution of tau proteins in the normal human central and peripheral nervous system.
Authors: Trojanowski J Q , Schuck T , Schmidt M L , Lee V M
Published: April 2011

In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.
Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo.
Authors: Schilling K , Duvernoy C , Keck S , Pilgrim C
Published: April 2011

We report that a monoclonal antibody directed against phosphorylated neurofilaments (SMI 31) recognizes nuclear antigens present in embryonic but not in adult neural cells. On Western blots, the antibody reacts with four proteins of apparent MW 35, 37, 52/54, and 250 KD which are found exclusively in developing brain tissue. These nuclear antigens are expressed by glial and neuronal cells. Both nuclear staining and immunoreactive proteins decrease with ongoing in vitro differentiation. A computer search for proteins that share the epitope recognized by antibody SMI 31 did not yield any proteins of known nuclear localization that exhibit the same molecular weights and solubility characteristics as the above immunoreactive proteins. We conclude that antibody SMI 31 recognizes hitherto unknown nuclear proteins which, in neural cells, are developmentally regulated.