bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural localization of daunomycin in multidrug-resistant cultured cells with modulation of the multidrug transporter.
Authors: Rutherford A V , Willingham M C
Published: April 2011

We localized the chemotherapeutic drug daunomycin inside cultured cells by taking advantage of its inherent fluorescence. Multidrug-resistant cultured cells, in which the accumulation of daunomycin can be reversibly controlled with verapamil to block the multidrug transporter, were incubated in daunomycin and verapamil and the accumulated daunomycin was visualized with epifluorescence optics. After fixation under a variety of different conditions to make cells permeable to diaminobenzidine (DAB), the internal daunomycin was illuminated under the fluorescence microscope in the presence of DAB. Photooxidation of DAB in sites of fluorescing daunomycin (photoconversion) resulted in intracellular deposition of oxidized DAB product. These sites were then visualized by transmission electron microscopy. In cells in which the multidrug transporter was inhibited by verapamil, daunomycin was localized in the nucleus of cells by mild fixation conditions such as formaldehyde. Increasing amounts of glutaraldehyde in the fixative caused apparent quenching of the nuclear fluorescence but still allowed fluorescence to occur in other cell organelles, which were then well preserved. Daunomycin was found in the nuclear envelope, the endoplasmic reticulum, and in lysosomes in cells in which the multidrug transporter was inhibited. Lysosomal accumulation has been previously described and was expected because of the known accumulation of positively charged molecules in organelles with low pH. However, the accumulation of daunomycin in the nuclear envelope and endoplasmic reticulum has not been previously observed. These results clearly demonstrate the utility of fluorescence photoconversion methodology for the high-resolution ultrastructural localization of fluorescent materials.
Immunogold labeling of calcium ATPase in sarcoplasmic reticulum of skeletal muscle: use of 1-nm, 5-nm, and 10-nm gold.
Authors: Dulhunty A F , Junankar P R , Stanhope C
Published: April 2011

We evaluated the use of immunogold electron microscopy to study the distribution of calcium ATPase in the sarcoplasmic reticulum membrane of skeletal muscle. We examined (a) 1-nm gold labeling, (b) the effect of gold size on immunolabeling, and (c) the densities of gold particles in areas of maximal labeling in fibers from rat extensor digitorum longus and pig gracilis muscles. The technique allowed unequivocal identification of the calcium ATPase. Gold particles of 1 nm were successfully visualized in unstained or lightly stained sections and the density of labeling was about 20 times greater than with 10-nm gold. The average densities in areas of intense labeling were 2878 +/- 139/microns 2 with 5-nm gold and 4310 +/- 276/microns 2 with 1-nm gold. These numbers are similar to the density of particles in freeze-fracture replicas of sarcoplasmic reticulum. The low density of 10-nm gold suggests that the large gold particles hinder binding of secondary to primary antibodies. The difference between 1- and 5-nm gold is explained by the amounts of gold conjugated to the immunoglobulin. The results suggest that there is a one-to-one relationship between secondary immunoglobulins (1-nm or 5-nm gold conjugates) and oligomeric complexes of calcium ATPase.
A specific fluorogenic reaction for tryptophan residues using isatin in organic solvents.
Authors: Trigoso C I , Ibañez N , Stockert J C
Published: April 2011

The development of new fluorogenic reactions for specific chemical groups is of increasing interest in cytochemistry. We describe the application of the nonfluorescent compound isatin on methanol-fixed blood smears. When treated with 0.1% isatin in absolute ethanol or acetone for 30 min and observed under violet-blue exciting light, eosinophil leucocyte granules show a bright green fluorescence. This fluorogenic reaction is abolished after blocking of tryptophan residues by performic acid oxidation. Spectrofluorometric studies with amino acids in vitro reveal that isatin forms a unique and specific fluorescent product with tryptophan.
Polysialosyl glycoconjugates defined by monoclonal antibody M6704 are expressed in human gliomas and embryonic neurons.
Authors: Nakayama J , Katsuyama T , Sugiyama E , Hirabayashi Y
Published: April 2011

The distribution of polysialosyl glycoconjugates defined by a monoclonal antibody M6704 (M6704 antigen) and of its O-acetylated counterpart (O-Ac-M6704 antigen) was investigated in human gliomas and in normal adult and fetal central nervous system. Among 32 gliomas, M6704 antigen was confined to the glioma cell surface and cytoplasmic processes in 21 cases, whereas O-Ac-M6704 antigen was expressed in the Golgi region in 22 cases and at the cell surface in seven cases. In adult tissues M6704 antigen was barely detectable, whereas in fetal tissues it was spatially and temporally expressed at the cell surface and along processes of post-mitotic neurons. The O-Ac-M6704 antigen first appeared in the Golgi regions of neurons at 66 gestational days and increased gradually with further maturation to adult levels. Several polysialoglycoproteins, the most dominant being 51 KD, and C-series polysialogangliosides were isolated from gliomas. These results indicate that M6704 antigen is a distinctive component expressed selectively by embryonic neurons during development and by glioma cells.
The mouse chimera during intrauterine stages: immunohistochemical analysis with the C3H strain-specific antibody.
Authors: Yoshiki A , Sakakura T , Kusakabe M
Published: April 2011

Our objective was to establish an immunohistological method for analysis of chimerism in mouse chimeras at embryonic stages with an anti-C3H strain-specific antigen (CSA) antibody. We developed an effective new method to retain CSA antigenicity with good morphology of embryonic tissues by using microwave irradiation (MWI) for pre-fixation, 95% ethanol/1% acetic acid as post-fixative solution, and polyester wax as embedding material. We used a biotinylated mouse monoclonal anti-CSA antibody, peroxidase-avidin, and silver amplification. These procedures were successful in demonstrating the chimerisms in various tissues of C3H<-->Balb/c chimeras at different embryonic stages and postnatal days. In chimeras at Days 7 and 7.5 post coitum (p.c.), both genotypes were clearly identified and well intermingling in every embryonic tissue (embryonic ectoderm, mesoderm, extra-embryonic ectoderm, ectoplacental cone, amnion, and chorion). Chimerisms at Day 14.5 p.c. were also clearly observed in mesencephalon, neural retina, spinal cord, lung, kidney, and liver. We concluded that the present immunohistological procedures for analysis of chimerism during embryonic periods will give us insightful information about dynamic histological changes such as cell proliferation, migration, selection, and death during organogenesis.
Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome.
Authors: Beil W J , Weller P F , Tzizik D M , Galli S J , Dvorak A M
Published: April 2011

Peripheral blood eosinophils from two normal donors and two patients with the hypereosinophilic syndrome (HES) were analyzed with a post-embedding immunogold method to detect the substructural location of tumor necrosis factor-alpha (TNF-alpha). In eosinophils of HES patients, TNF-alpha was localized to the matrix compartment of 64% of the specific secondary granules. Other structures in the HES eosinophils were unlabeled. No TNF-alpha was detected in eosinophils of normal donors. These studies document the first ultrastructural subcellular localization of any cytokine within the major population of secretory granules in human eosinophils and support other lines of evidence indicating that the expression of TNF-alpha may be greater in the eosinophils of HES patients than in those of normal donors.
Monoclonal antibodies to the Golgi apparatus of serous exocrine cells.
Authors: Yamashita S , Uchida H , Shiozawa M , Aiso S , Yasuda K
Published: April 2011

We demonstrated that a common antigen (Golgi-associated antigen, GAA 108) is present in the Golgi apparatus of serous exocrine cells, using an immunohistochemical method with monoclonal antibodies (MAb) 108 (IgG1) and 18 (IgM), raised to the microsomal fractions of rat parotid gland. The MAb reacted with polypeptides of molecular weights in the 58-170 KD range in parotid gland on Western blot analysis. The Golgi apparatus of the following cells was immunostained with these MAb: acinar cells of parotid gland, pancreas, and exorbital lacrimal gland, serous cells of sublingual gland, chief cells of stomach, and epithelial cells of rat prostate. However, positive reaction occurred throughout the entire cytoplasm of submandibular gland acinar cells. Immunoelectron microscopy (IM) revealed antigen (GAA 108) localization in the medial and trans-Golgi cisternae and trans-Golgi network (TGN), including condensing vacuoles, in parotid, exorbital lacrimal, and pancreatic acinar cells, and serous acinar cells of sublingual gland. Lysosomes and apical cell membranes also stained positively in some cells. In the submandibular gland reactions were observed in the medial and trans-Golgi cisternae, condensing vacuoles, secretory granule contents, cell membrane, and in some duct lumens. These results suggest that although GAA 108 is found in the Golgi apparatus of most serous exocrine cells, it is secreted by a regulated pathway in the acinar cells of submandibular gland.
Selective accumulation of diamidino yellow and chromomycin A3 by retinal glial cells.
Authors: Jeon C J , Masland R H
Published: April 2011

We applied the fluorescent DNA stains diamidino yellow (DY) and chromomycin A3 to rat and rabbit retinas in vivo and in vitro. They accumulated in the nuclei of a subpopulation of cells of the inner nuclear layer. The number and distribution of the fluorochrome-accumulating cells were similar to those of the Müller glia, and double-labeling experiments showed that the cells accumulating DY or chromomycin A3 contained oriented filaments of vimentin. The fluorochromes also accumulated in the sparse astrocytes and oligodendrocytes located among the myelinated fibers of the rabbit central retina. Specific accumulation in retinal glia occurred only when the fluorochromes were applied to living retinas. If the plasma membranes were disrupted by fixation or exposure to detergent, most retinal cells were stained. This indicates that the locus of specificity is the entry of the molecules into the cells. When applied to living retinas, other DNA stains selectively accumulate in subclasses of retinal neurons. Why DNA-binding molecules should selectively cross the membranes of either retinal neurons or retinal glia remains an unsolved problem.
Immunohistochemical localization of proteoglycans in human periodontium.
Authors: Häkkinen L , Oksala O , Salo T , Rahemtulla F , Larjava H
Published: April 2011

Proteoglycans (PGs) are extracellular and cell surface-associated macromolecules that regulate cell adhesion, cell growth, matrix formation, and bind growth factors. In this work we studied the distribution of core proteins of four PGs (decorin, biglycan, a large molecular weight PG, and CD44) in human gingiva and periodontal ligament by immunohistochemical staining of frozen tissue sections with specific antibodies. Decorin, a major PG of this tissue, was localized on collagen fiber bundles in the gingival and periodontal connective tissues. Staining for decorin was most intense at the subepithelial region. Biglycan was a minor PG component of the human periodontium, showing some accumulation in connective tissue under the oral epithelium. At the immunohistochemical level, biglycan appeared to form fine filament-like structures on extracellular matrix fibers. Localization of large molecular weight PG differed from that of decorin and biglycan. It was concentrated in deep connective tissue areas of the gingiva and in the periodontal ligament, and was only weakly present at the subepithelial region. CD44 was mainly concentrated in cell-cell contact areas of basal and spinous layers of oral epithelium. In the connective tissue of gingiva and periodontal ligament, CD44 was localized on fibroblast cell surfaces. Connective tissue area under the junctional epithelium contained relatively small amounts of PGs. The results indicate that different parts of human periodontium contain a typical variety of PGs, suggesting a specific function for each PG species in the location at which they accumulate.
Distribution of alpha 6 integrin subunit in developing mouse submandibular gland.
Authors: Kadoya Y , Yamashina S
Published: April 2011

We examined the development of mouse submandibular gland by light and electron microscopy and determined the distribution of the alpha 6 integrin subunit and laminin in this process by immunofluorescence and immunoelectron microscopy. At Days 13.5 and 14 of gestation alpha 6 was localized over the entire cell surface of undifferentiated epithelial cells of the terminal cluster. On Day 15 the expression of alpha 6 could no longer be detected over central cells in the proximal portion of branched terminal cluster, whereas peripheral cells were stained over the entire surface. On Day 17 of gestation to day of birth, alpha 6 expression was restricted to the basolateral surface of the differentiated acinotubular structure. Its expression on acinar cells was uniformly distributed throughout the basolateral membrane, but on duct cells stronger staining towards the basal surface was noted. Similar expression was observed in adult acinar and duct cells. Expression of the alpha 6-subunit at the cell-cell contact in an early stage and its expression at the basolateral surface in an advanced stage indicate that integrin containing alpha 6 plays a significant role in cell-cell and cell-substrate interaction. Stage and cell type-specific change of integrin expression may be significant for submandibular development.
Fundamental principles of in situ hybridization.
Authors: Wilcox J N
Published: April 2011

In situ hybridization provides invaluable information regarding the localization of gene expression in heterogeneous tissues. The technique is extremely sensitive and can detect the amount of mRNA contained in a single cell. This review provides a starting point for those who wish to begin using in situ hybridization in their own laboratories. The procedure outlined here is based on 35S-labeled riboprobes and has been used with many probes and tissues with a greater than 90% success rate on the first hybridization. The importance of appropriate controls is stressed. Clusters of silver grains after hybridization do not necessarily indicate specific mRNA localization. Regions of the tissue rich in nuclei often appear to cause spurious binding of probes and have high backgrounds often mistaken as positive signals. The most difficult aspect of in situ hybridization is not to get clusters of silver grains on the slide but rather to do the appropriate controlled experiments to ensure that the signal is real and is not due to some artifactual binding of the probe to the tissue.
A method for simultaneous detection of multiple mRNAs using digoxigenin and radioisotopic cRNA probes.
Authors: Miller M A , Kolb P E , Raskind M A
Published: April 2011

We present a simple, reliable method for simultaneous detection of two distinct mRNAs within the same tissue sample by double in situ hybridization histochemistry. Sections are hybridized with a cocktail of radiolabeled and digoxigenin-labeled cRNA probes. The digoxigenin-labeled probe is detected with an alkaline phosphatase (AP)-dependent chromogen reaction and then the radiolabeled probe is detected by conventional autoradiography. The sensitivities of the two detection methods are comparable and demonstrate the feasibility of using double in situ hybridization histochemistry to investigate the co-localization and co-regulation of mRNAs that are expressed at relatively low levels.
Estimation of tissue mRNAs by in situ hybridization.
Authors: McCabe J T , Bolender R P
Published: April 2011

We describe a new stereological method for analyzing data derived from the in situ hybridization procedure. This method should prove important, since data summarization in terms of grains per anatomic area by sampling of tissue sections may lead to faulty interpretations. Using computer simulation of measurements taken from a two- and a three-dimensional perspective, we show how the detection of molecular changes can be influenced by multiple structural events. Cell volume, the volume of the structure, and the number of labeled cells in an anatomic structure are all important parameters that must be assessed to obtain accurate results. We then outline in detail a simple and efficient method for estimation of the number of mRNA molecules in cells and in the total structure. By estimation of the abundance of mRNA molecules in three dimensions, we can make better inferences concerning gene expression in a structural context.
Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor.
Authors: Borke J L , Zaki A E , Eisenmann D R , Ashrafi S H , Ashrafi S S , Penniston J T
Published: April 2011

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.
Morphometric analysis of envelope glycoprotein gp120 distribution on HIV-1 virions.
Authors: Hart T K , Klinkner A M , Ventre J , Bugelski P J
Published: April 2011

The surface of HIV-1, like that of other retroviruses, is studied with virally encoded glycoproteins which appear ultrastructurally as electron-dense spikes or knobs. The glycoprotein that forms the spike structure, gp120, is non-covalently bound to the transmembrane glycoprotein gp41. Mature HIV-1 virions do not have as many spikes as the genetically related retroviruses HIV-2 and SIV. gp120 is lost from HIV-1 during viral morphogenesis and after incubation of the virus with the soluble form of cellular receptor CD4. In this study we used ultrastructural cytochemistry and morphometry to quantitate the distribution of envelope glycoprotein spikes on budding and mature HIV-1 virions and to look for alternatives to the laborious and somewhat subjective spike-counting technique for envelope spike analysis on HIV-1. HIV-1, strain HTLV-IIIB, was examined after staining of envelope glycoproteins with either tannic acid, immunogold staining for gp120 (gp120-immunogold), or lectin-gold staining with concanavalin A for mannose residues (ConA-HRP-gold) and frequency distributions of spikes or gold particles per micron HIV-1 membrane generated. Envelope spikes were normally distributed on membranes of budding and mature HIV-1. However, the density of spikes per micron viral membrane on mature HIV-1 virions was approximately 50% of that observed on budding virions. ConA-HRP-gold and gp120-immunogold did not efficiently label budding virions. The shape of the frequency distribution for ConA-HRP-gold particles on mature virions was similar to that for envelope spikes and could be used to quantitate envelope glycoproteins on HIV-1. In addition, ConA-HRP-gold staining was able to detect the loss of envelope proteins after treatment of virus with soluble CD4. gp120-immunogold labeling was patchy and many virions were unlabeled. ConA-HRP-gold staining proved to be a rapid, reliable, and easily quantifiable method for estimation of envelope glycoprotein density on mature HIV-1. However, the loss of spike structures throughout the life cycle of HIV-1 can effectively be determined only by direct spike counting.
Distribution of gamma-glutamyl transpeptidase in human salivary glands: immunohistochemical study using a monoclonal antibody.
Authors: Shiozawa M , Yasuda K , Yamashita S , Aiso S
Published: April 2011

We studied in detail the distribution pattern of gamma-glutamyl transpeptidase (gamma-GT) in human salivary glands using a monoclonal antibody (MAb) to human kidney gamma-GT. In the sublingual gland, a strong reactivity of the enzyme was recognized along the luminal and lateral membranes of serous cells. Weak but positive reactivity was noted on the luminal membrane of mucous cells. The intercellular canaliculi in the demilune and luminal surfaces of excretory duct cells were also immunoreactive. In the submandibular gland, a weak reaction was observed on the luminal membrane of intercalated duct cells and striated duct cells. Faint reactivity was seen on the luminal membrane of striated duct cells of the parotid gland. No reaction was observed in serous cells of the parotid gland. The immunoreaction in the sublingual gland was stronger than that in submandibular or parotid glands.
Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity.
Authors: Vankelecom H , Matthys P , Van Damme J , Heremans H , Billiau A , Denef C
Published: April 2011

We have previously shown that bioactive interleukin-6 (IL-6) is produced by rat and mouse (anterior) pituitary cells in vitro. Since the amount produced correlated with the presence of S-100-containing folliculostellate (FS) cells, these cells were suggested to be a source of IL-6 in the anterior pituitary (AP) lobe. In the present study we used immunocytochemical techniques to confirm this presumption. Freshly isolated mouse pituitary cells were subjected to immunocytochemical procedures whereby two different (neutralizing) monoclonal antibodies (MAb) against mouse IL-6 (6B4 and 20F3) and a polyclonal antiserum raised against bovine S-100 were used as primary antibodies. Single immunostaining revealed a small portion of mouse pituitary cells (about 6.5%) to be positive for IL-6 immunoreactivity with both antibodies. Importantly, the same proportion of cells was found to be IL-6 positive if only the AP was used as the cell source. About 7.5% of the pituitary cells stained for the presence of S-100 immunoreactivity. Positive staining for IL-6 was also found in pituitary cell samples from 2-day-old monolayer cultures and from redispersed 9-day-old histotypic aggregates, which both secreted bioassayable IL-6. In contrast, no IL-6 staining was found in AtT-20 cells, an established ACTH-secreting tumor cell line of the mouse pituitary which did not secrete bioactive IL-6. The specificity of the IL-6 immunostaining was demonstrated by a total loss of staining when MAb 6B4 was omitted or replaced by irrelevant rat IgG. Conclusively, pre-adsorption of the anti-IL-6 MAb (6B4) with recombinant mouse IL-6 totally abolished staining of pituitary cells. Double immunostaining for IL-6 and S-100 revealed that most if not all of the IL-6-containing pituitary cells were positive for S-100. Few of the S-100-containing cells did not stain for IL-6. These results confirm our previous hypothesis that FS cells, characterized by immunostaining of S-100 protein, contain bioactive and immunoreactive IL-6 and therefore are very likely producers of IL-6 in the AP. Furthermore, our results suggest that IL-6 is implicated in the local regulatory role ascribed to FS cells in the pituitary gland.
Sensitive detection of rat gastrin mRNA by in situ hybridization with chemically biotinylated oligodeoxynucleotides: validation, quantitation, and double-staining studies.
Authors: Larsson L I , Hougaard D M
Published: April 2011

Chemically biotin-labeled oligonucleotides form attractive reagents, as large quantities of stable and well-defined probes can easily be produced. Their usefulness for in situ hybridization was tested using rat gastrin cells as a model. Two probes recognizing two different regions of rat gastrin mRNA were synthesized and produced specific and equally strong hybridization signals. A probe complementary to human gastrin mRNA, but with mismatches to the rat gastrin mRNA sequence, failed to reveal rat gastrin cells under the stringency conditions used. Northern blotting revealed that the rat gastrin mRNA probes reacted exclusively with the appropriately sized (approximately 650 bases) mRNA. Model systems demonstrated that the hybridization signal, as revealed by alkaline phosphatase-based detection, varied linearly with the 10logarithm of target concentration and also showed that a new detection system was much more sensitive than previously used systems. In agreement with previous biochemical data, image analysis showed that starvation of rats led to a progressive decrease in cell staining intensities and cell numbers. Double staining for rat gastrin mRNA and gastrin immunoreactivity showed that in adult rats almost all gastrin cells expressed both mRNA and protein. Similar studies on developing rat gastrin cells revealed discrepancies between gastrin mRNA and gastrin-immunoreactive cells during the first week of newborn life. Subsequently, expression of mRNA and protein in the cells became gradually more concordant.
Localization of bone sialoprotein (BSP) to Golgi and post-Golgi secretory structures in osteoblasts and to discrete sites in early bone matrix.
Authors: Bianco P , Riminucci M , Silvestrini G , Bonucci E , Termine J D , Fisher L W , Robey P G
Published: April 2011

Bone sialoprotein (BSP), a bone matrix-enriched glycoprotein containing the Arg-Gly-Asp (RGD) motif and endowed with cell binding properties, was localized in osteoblasts and early bone matrix of developing rat bone at the ultrastructural level. Preliminary light microscopic observations indicated that intracellular labelling was restricted to a paranuclear dot corresponding to the "negative Golgi image" of classical histology. The same pattern was observed whether antisera against the fully glycosylated protein or a peptide antiserum to a stretch of amino acids in human BSP sequence were employed. At the EM level, we obtained labeling over the Golgi area of osteoblasts but not over the rER. The labeling was concentrated over distensions of the trans Golgi and over pro-secretory granules. In the matrix, BSP was distributed in a non-random manner. The label was concentrated over spherical aggregates of finely fibrillar material corresponding to the sites of early mineral deposition (so-called "mineralization nodules"). Such BSP-positive foci were seen both close to and away from the cell surface. The predominant association of BSP with Golgi and post-Golgi secretory structures and its absence from rER, as well as the reproducibility of the same pattern of localization with different antisera, might indicate a slow transit of the protein through the Golgi, not necessarily associated with protein glycosylation.
Distribution of the slow/cardiac isoform of skeletal muscle Ca(2+)-ATPase in developing and mature tissues of chickens determined using a monoclonal antibody.
Authors: Shahin S , Bartlett P F , Millar T J , McLennan I , Rostas J A
Published: April 2011

We have established that the monoclonal antibody (MAb) AA21, raised against a crude sarcolemmal fraction prepared from adult chicken anterior latissimus dorsi muscle, recognizes the slow twitch/cardiac isoform of calcium ATPase. This was done using a combination of immunohistochemistry at the light and electron microscopic level, the change in the cell distribution in skeletal muscle during development, the molecular weight of the principal protein recognized in Western transfers, and direct comparison with another MAb of known specificity. The antigen is initially expressed by all myotubes at E10 and with development is gradually lost from all presumptive fast fibers. In addition to its immunoreaction and slow extrafusal skeletal muscle fibers, AA21 displays a highly selective immunoreactivity with a number of other cell types in different tissues. The antibody stains a subset of intrafusal muscle fibers and intestinal and arterial smooth muscle, but not venous smooth muscle. In the nervous system, a subpopulation of neurons is intensely stained, most neurons are faintly stained, and glia are not stained at all.
A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ.
Authors: Huang Z , You W , Haugland R P , Paragas V B , Olson N A , Haugland R P
Published: April 2011

We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.
Bile canalicular contraction is coincident with reorganization of pericanalicular filaments and co-localization of actin and myosin-II.
Authors: Tsukada N , Phillips M J
Published: April 2011

We examined the relationships between actin-myosin interaction and bile canalicular contraction using a new experimental model: cytoskeleton-enriched canalicular membranes (CCM). In CCM, the bile canaliculus compartment is isolated complete with membrane-attached pericanalicular actin filaments and the surrounding intermediate filament sheath. Immunofluorescence and immunoelectron microscopy showed that actin and myosin-II were distributed over pericanalicular microfilaments that insert into adherens (belt) junctions; intermediate filaments predominantly inserted into desmosomes. The addition of "contraction solution" (1 microM Ca2+, 1 mM ATP) resulted in closure of CCM lumens, which was interpreted as canalicular contraction. Contraction was also associated with shortening and/or twisting of canaliculi. Rearrangement of actin filaments and myosin-II with co-localization of actin and myosin was observed. Evidence is also provided for attachment of actin-myosin-II aggregates to intermediate filaments coincident with contraction, suggesting a key scaffold function for intermediate filaments of the canaliculus. Attention is drawn to the overall similarity of structure-function dynamics in hepatic apical membranes to those described in intestinal brush border membrane preparations. The results are consistent with dynamic actin-myosin interaction with co-localization of actin and myosin-II in filament clumps coincident with canalicular contraction.
Immunoelectron microscopy of carbonic anhydrase isozyme VI in human submandibular gland: comparison with isozymes I and II.
Authors: Ogawa Y , Hong S S , Toyosawa S , Kuwahara H , Shimazaki M , Yagi T
Published: April 2011

Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.
Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development.
Authors: McCarthy K J , Bynum K , St John P L , Abrahamson D R , Couchman J R
Published: April 2011

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary basement membrane (GBM) but present in other basement membranes of the nephron, including collecting ducts, tubules, Bowman's capsule, and the glomerular mesangium. In light of this unique pattern of distribution and of the complex histoarchitectural reorganization occurring during nephrogenesis, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM-CSPG during nephrogenesis is unlike that reported for other basement membrane components such as laminin, fibronectin, and BM-HSPG, all of which can be found in the earliest formed basement membranes of the vesicle-stage nephron. Although BM-CSPG is present in the basement membranes of the invading vasculature and ureteric buds, its first appearance in nephron basement membrane occurs during the late comma stage. In capillary loop-stage glomeruli of prenatal animals, BM-CSPG is present in the presumptive mesangial matrix but undetectable in the GBM. However, as postnatal glomerular maturation progresses BM-CSPG is also found in both the lamina rara interna and lamina densa of the GBM in progressively increasing amounts, being most evident in the GBM of 21-day-old animals. Micrographs of glomeruli from 42-day-old animals show that BM-CSPG gradually disappears from the GBM and, by 56 days after birth, appears to be completely absent from the GBM, its pattern of distribution resembling that of the adult animal. Our results show that BM-CSPG is not required for the initial assembly of basement membranes but may in fact serve to stabilize basement membrane structure after histoarchitectural reorganization is completed.
Seasonal changes in testicular structure and localization of a sperm surface glycoprotein during spermatogenesis in sea urchins.
Authors: Ward R D , Nishioka D
Published: April 2011

Testes of the sea urchin, Strongylocentrotus purpuratus, undergo morphological changes consistent with a reproductive season that includes the winter months in the northern hemisphere (c. November to March). These changes can be observed in the basal germinal epithelia (BGE) of testicular acini as alterations in the gradients of meiosis and spermatogenesis from the peripheral (basal) to the inner (luminal) regions. Early in the season, large numbers of spermatogonia and primary spermatocytes along with large, spherical nutritive phagocytes (NPs) occupy most of the BGE. Relatively few secondary spermatocytes, spermatids, and spermatozoa are present. In the middle of the season more spermatocytes, spermatids, and spermatozoa are observed within well-defined spermatogenic columns that run between elongated NPs. Late in the season the BGE diminishes as the number of spermatogenic cells within it decreases dramatically and the lumen becomes distended with mature spermatozoa. To trace the origin and location of a 210 KD sperm surface antigen during spermatogenesis, a monoclonal antibody that recognizes this antigen, MAb J18/2, was used to probe testis sections. Indirect immunofluorescence microscopy detected the antigen throughout the BGE. Immunogold electron microscopy further localized it to the intracellular vesicles within spermatogenic cells of all stages. In mature spermatozoa the 210 KD surface antigen was detected not only on the sperm surfaces overlying the acrosomes and tails but also within the acrosome, suggesting the presence of an intracellular store of this antigen. Other than the occasional detection of antigen within the residual bodies of NPs, which contain phagocytosed spermatozoa, no antigen was detected in somatic cells of the testis, indicating that it is generated within the spermatogenic cells themselves.
Coexistence of renin and cathepsin B in secretory granules of granular duct cells in male mouse submandibular gland.
Authors: Sano K , Waguri S , Sato N , Kominami E , Uchiyama Y
Published: April 2011

Cathepsin B, a representative lysosomal cysteine proteinase, has been demonstrated to coexist with renin in secretary granules of rat pituitary LH/FSH cells and renal juxtaglomerular cells. We investigated immunocytochemically the localization of cathepsins B, H, and L in the submandibular gland of male mice, in which active renin is also produced. By light microscopy, granular immunodeposits for cathepsin B were detected in epithelial cells of the gland, particularly in granular duct cells and interstitial cells. Immunoreactivity for cathepsins H and L was mainly found in interstitial cells, although that for cathepsin H was weakly seen in acinar cells. By electron microscopy, immunogold particles indicating cathepsin B intensely labeled small granules near the Golgi complex of granular duct cells and weakly labeled large secretory granules, whereas those showing renin labeled both granules. Double immunostaining co-localized immunogold particles showing renin and cathepsin B in small perinuclear granules near the Golgi complex. Some immunopositive granules seemed to be closely associated with the Golgi elements. These results indicate that the co-localization of renin and cathepsin B is also seen in secretory granules of granular duct cells in the mouse submandibular gland, as seen in rat juxtaglomerular and LH/FSH cells. This suggests that cathepsin B is one of the possible candidates for the renin-processing enzyme.
Improvements in the silver-staining technique for nucleolar organizer regions (AgNOR).
Authors: Lindner L E
Published: April 2011

The silver-staining technique for nucleolar organizer regions (AgNOR) of Ploton et al., as popularized by Crocker, is being widely used for evaluation of nucleolar function, especially in neoplasia. It suffers from limited reliability, background staining, precipitates, and fading of the sections. Factors were identified that affect these problems. The oxidation-reduction level and gelatin used are particularly important. An improved procedure is presented which incorporates pre-reduction of the sections, selection of an optimal gelatin, and post-treatment of the sections to produce a permanent preparation. It is compatible with many fixatives and with other stains used before or after the silver stain.
Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section.
Authors: Trembleau A , Roche D , Calas A
Published: April 2011

We describe here a simple method for combining non-radioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a 35S-labeled oligonucleotide, and OT peptide in the same 12-microns cryostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBT/BCIP technique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin mRNAs, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues.
Renal tubule Na,K-ATPase polarity in glucocorticoid-induced polycystic kidney disease.
Authors: Ogborn M R , Sareen S , Grimm P C
Published: April 2011

Cyst formation in polycystic kidney disease (PKD) involves proliferation of cyst lining epithelial and changes in trans-epithelial fluid and electrolyte transport. In vitro studies have suggested that mislocation of Na,K-ATPase to the apical tubular surface may be an important component of cyst fluid transport. We undertook in vivo studies of Na,K-ATPase location using the "threshold" murine model of glucocorticoid-induced PKD (GIPKD). Using histological, immunohistochemical, and densitometric techniques, we compared cyst formation and the cellular location of Na,K-ATPase in suckling C3H (low threshold for GIPKD) and DBA (high threshold) mice given an inducing dose of 200 mg/kg methylprednisolone acetate. As expected, C3H mice demonstrated greater cyst formation as measured by proportion of section area occupied by the tubule lumen (26.7% vs 15.5%; p < 0.001). Cyst formation was associated with increased Na,K-ATPase staining and increased apical Na,K-ATPase location. MPA treatment in C3H mice resulted in apical staining that exceeded basolateral staining (35.3% of reference window vs 29.8%; p < 0.001). The relatively GIPKD-resistant DBA mice did not show such change in Na,K-ATPase location. These immunohistochemical studies suggest a role for Na,K-ATPase in renal cyst formation.
Immunocytochemical identification of cell types in benign and malignant breast diseases: variations in cell markers accompany the malignant state.
Authors: Rudland P S , Leinster S J , Winstanley J , Green B , Atkinson M , Zakhour H D
Published: April 2011

We performed immunocytochemical staining of benign, in situ, and malignant breast disease to identify antigens related to the presence of the major parenchymal cell types of the normal breast. Markers for the epithelial cells, antiserum to epithelial membrane antigen, and three monoclonal antibodies (MAb) to milk-fat globule membranes stained most of the inner cells in benign breast lesions, carcinoma in situ, and invasive carcinomas, but the peripheral cells in benign lesions, as well as in carcinoma in situ, were unstained. MAb to epithelium-specific keratin 18 stained the majority of inner cells in benign breast lesions but comparatively fewer such cells in carcinoma in situ and invasive carcinoma. Markers for the myoepithelial cells, antisera, and MAb to smooth muscle actin and vimentin stained most of the peripheral cells in benign breast lesions and in carcinoma in situ but failed to stain virtually any neoplastic cells in invasive carcinomas. Markers for the basement membrane adjacent to the myoepithelial cells, antiserum, and MAb to laminin and Type IV collagen delineated an intact basement membrane around benign lesions and carcinoma in situ, and fragmented structures in 5-10% of invasive carcinomas; the remaining carcinomas were largely unstained. Markers for both myoepithelial and epithelial cells, keratin MAb PKK2 and LP34, stained most of the inner cells in benign lesions but usually only relatively few malignant cells in carcinoma in situ and invasive carcinomas. Markers for the secretory alveolar cell, MAb to beta- and kappa-casein, stained a few isolated cells in benign lesions, many more inner cells in two such lesions in pregnant females, and none in invasive carcinomas. In conclusion, the myoepithelial cell and, under suitable hormonal conditions, the secretory alveolar cell, are retained in most benign lesions, but they are largely lost in invasive carcinomas.
Cholesterol ester in corpus luteum of rat observed by analytical color fluorescence electron microscopy.
Authors: Ning G , Fujimoto T , Koike H , Ogawa K
Published: April 2011

We used an analytical color fluorescence electron microscope to observe cathodoluminescence (CL) in the corpus luteum of rat. CL was emitted from lipid droplets and has a typical spectrum of two peaks at wavelengths of 320 and 430 nm. The intensity of CL at 320 nm (CL320) in the corpus luteum showed a regular change during an estrous cycle: it was very weak at the newly formed stage, gradually increased, reached the maximum at diestrus 2, and then began to diminish at proestrus except in the patches of degenerated cells. CL320 decreased during early stages of pregnancy or after prolonged treatment with 4-aminopyrazolo-pyrimidine; CL at 430 nm (CL430) remained clearly visible. CL320 showed a strong emission from degenerated luteal cells 10 days after hypophysectomy, but was diminished in cells rescued by injection of 50 IU pregnant mare's serum gonadotropin. In the luteal cells of luteinized ovary 2 hr after intravenous injection of 10 IU luteinizing hormone, CL was barely detected. CL320 in interstitial cells was also weak in the rats hypophysectomized and then treated with pregnant mare's serum gonadotropin, and in the 4-aminopyrazolo-pyrimidine-treated rats, although it has little change through a natural estrous cycle. The results are consistent with the assumption that the content of cholesterol ester is reflected by the intensity of CL320 emitted from the lipid droplets of rat luteal cells. The possibility was shown that the condition of steroidogenesis can be monitored through CL analysis by microscopy.