bluuebunny/crossref_metadata_2025 huggingface dataset:
Double labeled-antigen method for demonstration of intracellular antigens in paraffin-embedded tissues.
Authors: Falini B , De Solas I , Halverson C , Parker J W , Taylor C R
Published: April 2011

A double "labeled-antigen" method has been developed for the simultaneous staining of both kappa and lambda light chains in fixed paraffin sections. The method is a two step procedure utilizing a mixture of antisera against kappa and lambda light chains in the first stage, followed by the addition of a mixture of kappa antigen labeled with horseradish peroxidase and lambda antigen labeled with alkaline phosphatase. The selection of substrates yielding reaction products of contrasting color enabled the observer to distinguish kappa-containing cells (brown) from lambda-containing cells (blue). Reactive plasma cells stained either pure brown (kappa) or clear blue (lambda) in a ratio of 1.5:1. Blood vessels containing serum immunoglobulins showed a mixed brown-blue reaction, as did the Reed-Sternberg cells of some cases of Hodgkin's disease. The advantages of this double labeled-antigen method over previously reported methods for achieving double staining are discussed.
Immunocytochemical localization of kallikrein in the rat exocrine pancreas.
Authors: Bendayan M , Orstavik T B
Published: April 2011

The subcellular localization of kallikrein was studied in the rat pancreas using the immunocytochemical protein A-gold technique. Kallikrein was found at the level of the rough endoplasmic reticulum (RER), Golgi cisternae, condensing vacuoles, and zymogen granules of the pancreatic acinar cells as well as in the acinar lumen. The effect of various tissue processings on the immunocytochemical labeling of kallikrein was evaluated using pancreatic tissue fixed in glutaraldehyde and embedded in Epon, Lowicryl K4M, or glycol methacrylate (GMA). Compared to the results obtained with Epon, Lowicryl allowed improved resolution and specificity in the immunocytochemical labeling, while GMA retained greater amounts of kallikrein antigenicity leading to a higher intensity in the labeling; since it also gave a good ultrastructural preservation, GMA appeared to be the superior embedding medium for the localization of kallikrein. The quantitative evaluation of the labeling obtained under the three embedding conditions showed the presence of an increasing concentration gradient along the RER-Golgi-granule secretory pathway, suggesting that, like other pancreatic exocrine enzymes, kallikrein is synthesized in the RER, processed through the Golgi apparatus, and packed in the zymogen granules before being released into the acinar lumen.
An unlabeled antibody method using glucose oxidase-antiglucose oxidase complexes (GAG): a sensitive alternative to immunoperoxidase for the detection of tissue antigens.
Authors: Clark C A , Downs E C , Primus F J
Published: April 2011

Immunoenzyme staining for tissue antigens using glucose oxidase as marker enzyme was compared with immunoperoxidase techniques. Both the indirect, conjugated antibody method and the unlabeled antibody procedure employing preformed complexes of glucose oxidase-antiglucose oxidase (GAG) and peroxidase-antiperoxidase (PAP) were used to stain carcinoembryonic antigen (CEA) in paraffin tissue sections. The localization of CEA within a tissue specimen was the same in all cases, and background staining was minimal. The percentage of positive specimens detected with GAG and PAP was similar but was slightly greater with glucose oxidase compared to peroxidase conjugates. The glucose oxidase conjugates and GAG were similar to the comparable immunoperoxidase reagents in enzyme-antibody molar ratio, retention of antibody and enzyme activity, immunohistochemical staining dilutions, and stability. Immunoglucose oxidase and immunoperoxidase were combined to localize CEA and colon-specific antigen-p simultaneously. Excellent contrast and staining separation were shown between the enzymatic reaction products of the two systems. Since immunoglucose oxidase methods are as sensitive as the comparable immunoperoxidase techniques, they should be considered as a reliable alternative to the latter, especially when endogenous peroxidase activity may be a problem. Furthermore, the two methods can be conveniently combined for the simultaneous detection of two antigens.
Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry.
Authors: Hsu S M , Soban E
Published: April 2011

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.
Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. I. Ultrastructure and phosphatase activity of endodermal cells.
Authors: Laurie G W , Leblond C P
Published: April 2011

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.
Immunochemical localization of metallothionein in rat liver and kidney.
Authors: Danielson K G , Ohi S , Huang P C
Published: April 2011

An indirect immunoperoxidase staining technique was employed to localize the heavy metal binding protein, metallothionein, in rat liver and kidney. Immunostaining for metallothionein was observed in all hepatocytes and most renal tubular cells. This protein was not detectable, however, in cell types such as endothelial or connective tissue cells, indicating that metallothionein synthesis or accumulation is tissue- and cell-type specific. Hepatocytes from cadmium-exposed animals showed increased staining for metallothionein. The presence of metallothionein was also seen extracellularly within the liver sinusoids and renal tubules in both normal and cadmium-exposed animals, suggesting transport and excretion, respectively, of this protein.
Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. II. Immunostaining for type IV collagen and its precursors.
Authors: Laurie G W , Leblond C P , Martin G R
Published: April 2011

Antibodies to type IV collagen were linked with peroxidase and used for direct immunostaining of Reichert's membrane and the associated cells of the rat parietal yolk sac. Immunostaining was observed throughout the thickness of Reichert's membrane and within the endodermal cells arranged as a single layer on its inner side. The immunostaining of endodermal cells was mainly present in the cisternae of rough endoplasmic reticulum (rER) and in the Golgi apparatus, where it could occur in any saccule, but predominated in the GERL elements and associated prosecretory granule-like structures. Moreover, the secretory granule-like structures present in the ectoplasm next to Reichert's membrane were also immunostained. Finally, immunostaining was observed in multivesicular bodies and occasionally in secondary lysosomes. The antigenicity detected by immunostaining in Reichert's membrane is attributed to type IV collagen itself, whereas the antigenicity of endodermal cells is assigned to precursors of this collagen. It is proposed that initial precursors arise in rER cisternae, migrate to Golgi saccules, and pass to the GERL element, where they accumulate into prosecretory granules, which, perhaps by fusion with one another, become secretory granules. The secretory granules in turn migrate to the cell surface where they release their content, which becomes the type IV collagen of Reichert's membrane. Some diversion from this pathway may account for the immunostaining of multivesicular bodies and lysosomes.
Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. III. Immunostaining for laminin and its precursors.
Authors: Laurie G W , Leblond C P , Martin G R , Silver M H
Published: April 2011

Reichert's membrane and the endodermal cells of the parietal yolk sac were examined for the presence of laminin antigenicity using anti-laminin antibodies and the peroxidase-antiperoxidase sequence. Immunostaining was observed through the full width of Reichert's membrane and within endodermal cells. In these cells immunostaining was observed in rough endoplasmic reticulum (rER) cisternae and Golgi apparatus. The Golgi staining could occur in any saccule, but predominated in components interpreted as the last saccule of the stack, the GERL element, and associated prosecretory granules. The secretory granules found in the ectoplasm were also immunostained. Finally, multivesicular bodies showed some staining. The immunostaining of Reichert's membrane indicates the presence of laminin itself, while that of rER cisternae and the Golgi apparatus is attributed to laminin precursors. Presumably the biosynthesis of laminin occurs along the usual protein pathway, that is, from rER through Golgi saccules and the GERL element to secretory granules, which release their content into Reichert's membrane. The laminin immunostaining of Reichert's membrane and endodermal cells is similar to that of type IV collagen. It is, therefore, likely that the two substances are processed and secreted simultaneously.
Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method.
Authors: Mitani F , Shimizu T , Ueno R , Ishimura Y , Izumi S , Komatsu N , Watanabe K
Published: April 2011

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.
Carbonic anhydrase in the type I skeletal muscle fibers of the rat. An immunohistochemical study.
Authors: Väänänen H K , Kumpulainen T , Korhonen L K
Published: April 2011

The localization of carbonic anhydrase (CA) was studied in rat skeletal muscles with the use of immunohistochemical (peroxidase-antiperoxidase) method. CA was observed in all those fibers that also showed pH 4.3 stable actomyosin adenosine triphosphatase activity (type I fibers), but the reverse did not necessarily hold. More specifically, CA was apparently localized in I-bands, and a weak reaction was also observed in sarcolemma. The function of CA in muscle fibers is possibly connected with the greater demands on CO2 transport and buffer system in muscles adapted to long-lasting contractions.
Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands.
Authors: Orstavik T B , Carretero O A , Hayashi H , Scicli G A , Johansen L
Published: April 2011

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.
Adenylate cyclase localization in unfixed specimens of rat oral mucosa and isolated mitochondria.
Authors: Fine A S , Egnor R W , Forrester E , Stahl S S
Published: April 2011

The cytochemical localization of adenylate cyclase (E.C.4.6.1.1) was demonstrated in unfixed rat oral mucosal specimens and isolated mitochondrial fractions by use of a modified cytochemical reaction mixture. The adenylate cyclase reaction product was observed in the mitochondrial cristae of intact epithelial and fibroblast cells, and in isolated mitochondria after a 10-min incubation in the reaction mixture. The validity of the cytochemical adenylate cyclase method was supported by biochemical studies.
Ultrastructural localization of the low molecular weight protease inhibitor in human bronchial glands.
Authors: Mooren H W , Meyer C J , Kramps J A , Franken C , Dijkman J H
Published: April 2011

Light microscopically the low molecular weight protease inhibitor (LMI) and lysozyme have both been demonstrated in the cytoplasm of serous cells of bronchial glands. By immunoelectron microscopy LMI was demonstrated in secretory granules of these serous cells. Many granules showed a peripheral or bean-shaped staining pattern, other granules were uniformly stained or not stained at all. In contrast to this latter finding, virtually all granules were found positive for the presence of lysozyme, suggesting that there is no association of LMI with lysozyme at the ultrastructural level.
Electron microscopic localization of specific carbohydrate groups in thin sections of tissues embedded in a hydrophilic resin: concanavalin A receptors in mouse spleen.
Authors: Shida H , Shida M , Gorbsky G , Steinberg M S
Published: April 2011

An improved postembedding method for electron microscopic localization of concanavalin A (Con A) receptors in ultrathin sections is reported. Materials are embedded in a hydrophilic resin, glycol methacrylate copolymerized with glutaraldehyde and urea, using a precisely controlled polymerization schedule that preserves ultrastructural integrity. Ferritin-Con A binds specifically in ultrathin sections of mouse spleen tissue and of Sephadex beads embedded in this resin. Quantitative investigations of procedures for decreasing nonspecific labeling demonstrate that preincubation of sections with bovine serum albumin reduces background to a very low level. This high-resolution method allows macromolecular affinity probes access to receptors in all cellular locations, while maintaining good morphological preservation.
Enzyme histochemical studies on the pathological changes in human Sertoli cells.
Authors: Goslar H G , Hilscher B , Haider S G , Hofmann N , Passia D , Hilscher W
Published: April 2011

Two forms of human Sertoli cell disorders were characterized enzyme histochemically from the testicular biopsy material of infertile and subfertile patients. Sertoli cell asthenia: a slight injury of the Sertoli cell with exfoliation of individual germ cells; marked by the rarefaction of reaction zones of thiamine pyrophosphatase (TPPase) and a decrease in lactate dehydrogenase (LDH). Sertoli cell insufficiency: severe Sertoli cell damage with the formation of a "puff" and a heavy exfoliation of germ cells (dislocation of Sertoli cell nucleus and cytoplasm along with the related germ cells into the lumen of seminiferous tubule); marked by a heterogeneous activity pattern of TPPase, the disappearance of LDH, maintenance of a slightly weakened activity of alkaline phosphatase, and an increase of acid phosphatase. In the case of Sertoli-cell-only syndrome, the high prismatic Sertoli cells showed strong acid phosphatase activity with scattered weak TPPase reaction, whereas the flat or cube-like Sertoli cells exhibited weak acid phosphatase activity with only one small round reaction zone of TPPase in each cell. In addition, the frequency of the occurrence of Sertoli cell asthenia, Sertoli cell insufficiency, and Sertoli-cell-only syndrome is reported, and its correlation with the andrological diseases discussed.
Localization of renal and intestinal trehalase with immunofluorescence- and enzyme-labeled antibody techniques.
Authors: Nakano M
Published: April 2011

The localization of trehalase with fluorescein isothiocyanate-conjugated and peroxidase-conjugated antibody techniques was examined. Antiserum against purified rabbit renal trehalase was produced against guinea pigs. Anti-renal trehalase immunoglobulin (Ig)G was isolated from the serum and used for the immunohistochemical localization of intestinal and renal trehalases. Specific fluorescence and peroxidase staining were observed in the brush borders of proximal tubules and of intestinal epithelial cells. These results are in good agreement with the biochemical results. Thus, it is concluded that trehalase is specifically localized in the renal and intestinal brush borders. Sections of rabbit intestine and of rabbit kidney treated with anti-rabbit renal trehalase IgG were observed to have a specific fluorescence at the brush borders. Sections of rat intestine treated with the same antibody, however, showed no specific fluorescence at the brush borders. From these results, it is strongly suggested that renal trehalase and intestinal trehalase from the rabbit have common antigenic determinants and that these differ from those in rat intestinal trehalase.
Membrane histochemistry of Physarum polycephalum myxamoebae.
Authors: Aldrich H C , Reiskind J B
Published: April 2011

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.
Measurement of tissue disulfide groups by Alcian blue 8GX and atomic absorption spectrophotometry.
Authors: Rosenquist T , Stewart D
Published: April 2011

The primary purpose of this study was to quantify tissue disulfides with Alcian blue 8GX using atomic absorption spectrophotometry (ASS) to measure the copper moiety of the Alcian blue. The secondary purpose was to determine whether aqueous or alcoholic formalin was a better fixative for this procedure. Sections of mouse renal cortex fixed in 10% neutral formalin (NBF) or in ethanol/formalin/acetic acid (AFA) were stained in 0.5% Alcian blue 8GX in 0.8 M MgCl2 with prior thiosulfation (+Th) or without prior thiosulfation (-Th). The relative contents in the tissues of the copper of Alcian blue, measured by AAS, may be summarized as follows: -Th NBF, 1.15 micrograms Cu/mg tissue; -Th AFA, 1.35 micrograms Cu/mg tissue; +Th NBF, 1.99 micrograms Cu/mg tissue; +Th AFA, 3.25 micrograms Cu/mg tissue. These data indicate that 1) disulfide may be measured by this procedure, and 2) AFA is the preferred fixative for measurement of both disulfides and native sulfates.
Lanthanum as an electron microscopic stain.
Authors: Shaklai M , Tavassoli M
Published: April 2011

Applications of lanthanum as an electron microscopic tracer have been reviewed. This electron-dense trivalent cation, which binds avidly to calcium binding sites, can be used as tracer for delineating extracellular spaces and intercellular junctions. It has served as a basis for classification of junctional structures. It can also be used as a calcium probe, a tracer in studying the permeability of barriers, as an intracellular marker and as an electron microscopic stain for such membrane components as surface glycoprotein. Each of these applications may require a different methodology. Thus methodological considerations in the use of this tracer have also been reviewed. The recent recognition that lanthanum is more than a passive tracer and that by reacting with different cell components may serve as a true stain, will extend the use of lanthanum in electron microscope histochemistry.
Application of a rapid avidin--biotin--peroxidase complex (ABC) technique to the localization of pituitary hormones at the electron microscopic level.
Authors: Childs G V , Unabia G
Published: April 2011

The new avidin--biotin--peroxidase complex (ABC) technique was applied to ultrathin sections of rat pituitary that were fixed with glutaraldehyde and embedded in Araldite 6005. The primary antisera dilutions that are normally applied for 24-48 hr with the peroxidase-antiperoxidase (PAP) complex technique were used. High background was observed with the ABC method when incubation times were 12-48 hr. Tests were then conducted with shorter incubation times. The staining intensity was measured with a densitometer. Detectable stain was seen after only 15 min in dilutions of 1:10,000 anti-bovine luteinizing hormone (bLH beta), 1:8000 anti-rat thyroid-stimulating hormone (rTSH beta), and 1:20,000 anti-25-39-adrenocorticotropic hormone (25-39ACTH). Optimal LH staining was seen after 30 min, whereas optimal staining for TSH or ACTH required 1 hr. Stain was detectable with a dilution of 1:4000 anti-human follicle-stimulating hormone (hFSH beta) after 30 min and was optimal after 4 hr. Prolonged incubation times with these dilutions decreased the staining intensity because a deposit of high background was produced that appeared as a filigreed network over the cells. When higher dilutions were tested with 2-hr incubation times, optimal staining was seen with 1:30,000 anti-bLH beta, 1:24,000 anti-rTSH beta, 1:30,000 anti-25-39ACTH, and 1:8000 anti-hFSH beta. These tests demonstrate the potential of the ABC method for the rapid detection of small amounts of specific and nonspecific antibodies that are bound to pituitary cells.
Surgical and intensification procedures for defining visual pathways with cobaltous-lysine.
Authors: Springer A D , Prokosch J H
Published: April 2011

The introduction of heavy metals directly into living neuronal tissue has received little attention as a method for defining connections in the vertebrate nervous system. Procedures are described for the use of cobaltous-lysine to trace visual pathways with light microscopy. The success of the technique depends on a number of variables including: the method by which the tracer is applied, the survival period, and the method of intensification. Best results were obtained by soaking the optic nerve of living animals in the filling solution for 24 hr and by using a modification of the Timm's silver sulfide intensification procedure. Several factors that lead to poor results are discussed. Cobaltous-lysine is capable of both anterograde and retrograde labeling and can fill very sparse projections. Furthermore, the success of the fill can, at times, be evaluated prior to histology.
A cytological approach to the role of guanine in determining quinacrine fluorescence response in eukaryotic chromosomes.
Authors: Ferrucci L , Mezzanotte R
Published: April 2011

Photooxidation is believed to preferentially remove guanine (G) residues from chromosomal DNA. G interspersion, moreover, has been hypothesized as quenching quinacrine (Q) fluorescence in cytological preparations. Hence, we used photooxidation as a tool for inducing possible changes in the Q-banding pattern of Drosophila melanogaster, Drosophila virilis, and Mus musculus metaphase chromosomes. An enhanced Q fluorescence, which was particularly evident in certain chromosomal regions, was found. This finding would support the postulated primary role of G in determining Q bands in eukaryotic chromosomes.
Acrolein/glutaraldehyde as a fixative for combined light and electron microscopic immunocytochemical detection of pituitary hormones in immersion-fixed tissue.
Authors: Smith P F , Keefer D A
Published: April 2011

In an attempt to find a fixative that has a wide range of application in immunocytochemical studies of pituitary tissue, we have investigated the use of acrolein fixation followed by postfixation in osmium. Pituitary glands were excised and immersion-fixed in 2 or 5% acrolein/0.25% glutaraldehyde for 1, 2.5, 5, or 24 hr. Some tissues were subsequently postfixed in 1% OsO4 for 1.5 hr before embedding in Epon-Araldite. Sections were collected for both light and electron microscopic immunocytochemistry, employing several pituitary hormone antisera. Fixation for 2.5 hr with 2% acrolein/0.25% glutaraldehyde followed by osmication resulted in good staining for both light and electron microscopy with all antisera employed. Furthermore, the rapid penetration qualities of this fixative resulted in good ultrastructural preservation throughout the tissue.
A general method for heterokaryon detection using resonance energy transfer and a fluorescence-activated cell sorter.
Authors: Wanda P E , Smith J D
Published: April 2011

A simple and general labeling method has been adapted for the rapid detection and isolation of heterokaryons soon after somatic cell fusion. The method involves prelabeling each parental cell type by adding one of two hydrophobic fluorescent probes, F18 or R16, to the culture medium. These probes are nontoxic, nonmutagenic, and do not inhibit either cellular replication or the efficiency of fusion. Following polyethylene glycol (PEG)-induced fusion, heterokaryons are identified on a cell sorter as cells showing fluorescence as a result of nonradiative resonance energy transfer (RET) between the two fluorochromes. Optimal conditions are described for the unambiguous detection of heterokaryons by fusion-induced RET. The value of this method lies in the universal applicability of F18 and R16 as simple and direct membrane labels for any pair of parental cells, even those lacking selectable genetic markers or detectable antigenic differences. This potential for heterokaryon selection in any cell cross should significantly expand the range of cell types amenable to investigation through somatic cell genetics, while the rapidity of the method should facilitate the study of early events following fusion.
Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.
Authors: Dudek R W , Childs G V , Boyne A F
Published: April 2011

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.
Versatility of biotin-labeled lectins and avidin-biotin-peroxidase complex for localization of carbohydrate in tissue sections.
Authors: Hsu S M , Raine L
Published: April 2011

Carbohydrate residues in tissue can be localized by a very sensitive, specific, and simple technique, utilizing a biotin-labeled lectin followed by an avidin-biotin-peroxidase complex (ABC). Distribution of peanut receptors in benign and malignant tissues was found to be quite different. Normal colonic mucosa occasionally shows small dot staining in the supranuclear cytoplasm, while the tumor cells of colonic adenocarcinoma tend to show diffuse cytoplasmic or membranous staining. Normal breast ductal epithelium shows membranous staining at luminal borders, while medullary carcinoma of the breast shows distinct membranous and cytoplasmic staining. The use of this avidin-biotin-peroxidase complex technique may contribute to understanding the process of tumorigenesis as well as providing a sensitive method to determine early stages of malignant transformation.
Cytotoxicity, Mutagenicity and DNA damage by Hoechst 33342.
Authors: Durand R E , Olive P L
Published: April 2011

Hoechst 33342 can be used for flow microfluorimetry (FMF) analysis of the DNA content of living Chinese hamster cells, giving good resolution (coefficients of variation (CVs) 6%) with relatively nontoxic staining regimens. The dye is, however, a very efficient inhibitor of DNA synthesis, with a marked depression of the DNA synthetic rate for V79 cells observed at concentrations tenfold less than those required for optimal FMF resolution. Nontoxic and minimally toxic Hoechst concentrations also resulted in demonstrable mutation, as assayed by 6-thioguanine resistance. Hoechst disappearance from cells returned to normal growth medium after staining suggests two components of binding, since about half of the total stain is rapidly removed, whereas the rest is apparently diluted only by cell division. Cells containing Hoechst 33342 die more rapidly than control cells when held at 4 degrees C and are also more susceptible to inactivation by the ultraviolet laser beam if operated at approximately greater than 100 mW power. Thus, Hoechst 33342 can provide information about the DNA content of living cells, but at the expense of minimal toxicity, moderate mutation, and significant cell cycle perturbations.
High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry.
Authors: Gallyas F , Görcs T , Merchenthaler I
Published: April 2011

A simple and reliable method is described for the intensification of the end-product of the diaminobenzidine reaction demonstrating peroxidase activity. After completing the diaminobenzidine reaction, the preparations to be intensified are immersed first in thioglycolic acid solution, then in distilled water, and finally in a special physical developer containing silver nitrate.
Location and chemical composition of anionic sites in Bruch's membrane of the rat.
Authors: Pino R M , Essner E , Pino L C
Published: April 2011

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.
Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat.
Authors: Dixon R G , Eng L F
Published: April 2011

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.
Tissue localization of lymphocyte surface antigens and receptors for immunoglobulin G Fc and complement in the chicken.
Authors: Thunold S , Boyd R , Schauenstein K , Wick G
Published: April 2011

Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.
Metastatic tumor cell attachment and invasion assay utilizing vascular endothelial cell monolayers.
Authors: Nicolson G L
Published: April 2011

Two of the more important steps in blood-borne tumor metastasis are attachment of the circulating malignant cells to the vascular endothelium and subsequent extravasation or invasion out of the blood vessel. A model for this process has been developed using cultured monolayers of vascular endothelial cells that synthesize a basal lamina or extracellular matrix (Kramer and Nicolson, Proc Natl Acad Sci USA 76:504, 1979). We have used this model to study metastatic tumor cell-endothelial cell interactions such as attachment to endothelial cells and their subsequent retraction and exposure of endothelial basal lamina as well as the interactions of metastatic tumor cells with the basal lamina leading to invasion and solubilization of this extracellular matrix. Morphological, immunological, and enzymological analysis of these steps in the metastatic process can be obtained using the vascular endothelial cell monolayer model for attachment and invasion.
The histochemical demonstration of gamma-glutamyl transpeptidase activity in different populations of rat liver during azo dye carcinogenesis.
Authors: Daoust R
Published: April 2011

To better assess the reliability of gamma-glutamyl transpeptidase (gamma-GTase) as a marker of preneoplastic liver lesions and hepatomas, the gamma-GTase activity of different cell populations was examined in liver sections from rats fed 4-dimethylaminoazobenzene. The results indicated that the biliary ductular cells in trabeculae of cirrhotic livers may exhibit appreciable gamma-GTase activity in addition to that shown by islands of regenerating parenchyma. At later stages of azo dye carcinogenesis, the epithelial cells of bile duct cysts and cholangiomas, as well as those of hepatomas, gave positive reactions for gamma-GTase. Thus biochemical data on liver gamma-GTase in different models of hepatocarcinogenesis cannot be translated directly in terms of alterations in a particular cell type unless such interpretation is justified by parallel histochemical investigations.
Ultracytochemical localization of estrogen-stimulated guanylate cyclase in rat uterus.
Authors: Kang Y H , Sahai A , Criss W E , West W L
Published: April 2011

Estrogens are known to increase cyclic guanosine monophosphate (cGMP) levels in the uterus of rats by enhancing guanylate cyclase (GC) activity. In the present study, the cytochemical localization of GC activity was studied in the uteri of immature and ovariectomized rats after treatment with diethylstilbestrol (DES), progesterone, estrogen antagonist (CI628), and a combination of DES and CI628. Twenty-four hours after the first dose of DES, moderate to strong guanylate cyclase activity was indicated by lead phosphate precipitate on the luminal microvillar and basolateral surfaces of epithelial cells, whereas strong activity was found on the plasma membranes of fibroblasts, endothelial cells, and myometrial cells. The enzyme activity in the epithelial cells declined slightly 24 hr after the second daily dose of DES. Uterine tissues from DES-treated rats that were preheated at 60 degrees C for 30 min or preincubated with a GC inhibitor showed no reaction product. Guanylate cyclase activity was not observed cytochemically in the uterine tissues of the vehicle control (immature or ovariectomized) or progesterone-and CI628-treated animals. Weak guanylate cyclase activity was observed on the plasma membranes of epithelial cells and endothelial cells after doses of DES and CI628 were given simultaneously. The biochemical assays of the total homogenate in vitro indicated that uterine GC showed about a twofold increase after one dose of DES and a 1.3-fold increase following two doses (one dose per day) of DES when compared with their respective nontreated controls, or with progesterone-treated uteri. GC was found in particulate (09%) and cytosol (10%) fractions. These data demonstrated that DES stimulated uterine guanylate cyclase activity, while progesterone and CI628 were ineffective at the doses used. Estrogen antagonist CI628 doses not completely suppress the effect of DES.
Immunohistochemical localization of cyclic guanosine monophosphate (cGMP) on mouse fetal chromosomes.
Authors: Tucci S M , Rosenberg E M
Published: April 2011

In previous immunohistochemistry studies, cyclic guanosine monophosphate (cGMP) has been found in polytene chromosomes of D. melanogaster, cGMP has not been found in mammalian metaphase chromosomes, but this could be due to loss of cGMP during staining. Thus the effect of different fixation techniques on the immunohistochemically detectable cGMP associated with metaphase chromosomes from mouse fetal tissue was examined. In chromosomes from cells fixed in 2% formalin, or unfixed cells dropped on slides preheated to 60 degrees C, there was diffuse cGMP staining. When cells were fixed in methanol:glacial acetic acid, 3:1, no chromosomal cGMP immunofluorescence was observed, whereas chromosomes from cells fixed in methanol:glacial acetic acid, 6:1, had different patterns of cGMP immunofluorescence depending on the temperature of the slides onto which the fixed cells were dropped. On slides prechilled to 4 degrees C, cGMP immunofluorescence outlined the chromosomes; on room temperature slides, faint chromosomal cGMP staining was observed, and on slides preheated to 68 degrees C or room temperature slides blown dry with hot air, the chromosomes had more intense diffuse cGMP immunofluorescence or distinct symmetrical bands of cGMP immunofluorescence. We have demonstrated the presence of cGMP in mammalian metaphase chromosomes. The different patterns of cGMP immunofluorescence observed may reflect variable preservation of chromosomal proteins that have binding sites for cGMP.
A modified glyoxylic acid-formaldehyde technique for histofluorescence of catecholamine-containing neurons in cryostat sections of the insect brain.
Authors: Klemm N
Published: April 2011

A simple and rapid method is described to demonstrate catecholamine-containing neurons in the insect brain by cryostat sectioning and immersion in an ice-cold SPG (sucrose-potassium phosphate-glyoxylic acid) solution. The fluorescent yield achieved can be further intensified by heating the preparations with formaldehyde vapors. The advantages of the method as compared to the original Falck-Hillarp method are speed, simplicity, and a very distinct photostabile fluorophore. The preparations can be stored for several weeks. The drawback is a lower sensitivity than the Falck-Hillarp method provides.
DNA dependence and inhibition by novobiocin and coumermycin of the nucleolar adenosine triphosphatase (ATPase) of human fibroblasts.
Authors: Fox N , Studzinski G P
Published: April 2011

We have recently demonstrated by electron microscopic cytochemical methods that unfixed human fibroblasts exhibit intense MG2+ dependent adenosine triphosphatase (nATPase) activity in circumscribed areas of the cell nucleoli. The nATPase was specific for ATP and dATP and was inhibited by other ribonucleoside triphosphates. Its intranucleolar localization relative to nucleolar chromatin, and segregation into nucleolar zones after actinomycin treatment of the cells, suggested that the reaction took place in fibrillar centers. This ATPase has now been further characterized by electron microscopic cytochemistry. It was determined that short fixation permitted retention of most of the ATPase activity, and that the enzyme was active at high ionic strength (up to 400 mM KCl), but that the enzyme activity was very sensitive to elevated temperatures. DNA dependence of the enzyme was shown by inhibition of the reaction by DNase pretreatment in parallel with the removal of DNA from the cell, while pretreatment with RNase had no significant effect. The nATPase activity was also selectively inhibited by treatment of the cells with antagonists of the B subunit of DNA gyrase, novobiocin, and coumermycin, but not by nalidixic or oxolinic acids, which interfere with the A subunit of gyrase. Inhibitors of RNA synthesis, actinomycin D and aminonucleoside of puromycin, potentiate rather than inhibit nATPase reaction. The results suggest that nATPase functions to alter the degree of supercoiling or catenation of nucleolar organizer DNA, and is in reality a DNA topoisomerase that hydrolyzes ATP during its action.
Specificity and sensitivity of immunohistochemical detection of factor VIII/von Willebrand factor antigen in formalin-fixed paraffin-embedded tissue.
Authors: McComb R D , Jones T R , Pizzo S V , Bigner D D
Published: April 2011

The immunohistochemical detection of factor VIII/von Willebrand factor antigen (FVIII/vWF-AG) in formalin-fixed paraffin-embedded tissues was investigated using the peroxidase-antiperoxidase (PAP) method. Highly purified human FVIII/vWF was used to raise rabbit anti-FVIII/vWF-AG serum. In addition to anti-FVIII/vWF-AG activity, the unabsorbed antiserum had anti-IgG, anti-IgM, and anti-alpha2-macroglobulin specificities. Following exhaustive absorption with these proteins, the antiserum reacted monospecifically for FVIII/vWF-AG in immunodiffusion, immunoelectrophoresis, and PAP immunohistochemistry. Sections of normal tissues from six patients and a total of 43 neoplasms were examined. Treatment of the tissue sections with trypsin prior to application of the antiserum markedly increased the sensitivity of FVIII/vWF-AG detection. The positive staining for FVIII/vWF-AG was restricted to endothelial cells in both neoplastic and nonneoplastic tissue. In general, the hyperplastic endothelia in neoplastic and reactive tissues stained more intensely than those in normal tissues. Expression of FVIII/vWF-AG by nonendothelial neoplastic cells was not observed. FVIII/vWF-AG is a reliable marker for endothelial cells.