bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium.
Authors: Farr A , Nelson A , Truex J , Hosier S
Published: April 2011

A monoclonal antibody (MAb), G8.8, was raised against glycoconjugates isolated from a cloned line of murine medullary thymic epithelial cells. Flow cytometric analysis of the reactivity of this MAb with cultured thymic epithelium demonstrated that the ligand was expressed on the cell surface. Immunohistochemical examination of normal murine thymus revealed labeling of cells in the subcapsular and medullary areas. Immunoelectron microscopy revealed surface labeling restricted to cells possessing ultrastructural features of epithelium (desmosomes, tonofilaments, and cytoplasmic cysts). During thymic ontogeny, G8.8+ cells predominated in fetal development at the earliest time point examined (Day 14 of gestation). There was an expansion of the cortical epithelial component so that by Day 18 cortical and medullary compartments could be clearly distinguished. Immunoprecipitation of radioiodinated thymic stroma with MAb G8.8 detected a molecule with an apparent Mr of approximately 38 KD under non-reducing conditions. When reduced, the apparent Mr was slightly increased (42 KD). This MAb also exhibited reactivity with gut and epidermal epithelium and some tubular epithelium in the kidney, but did not react with epithelial parenchymal cells of the liver.
Location of glandular kallikrein mRNAs in mouse submandibular gland at the cellular and ultrastructural level by hybridization histochemistry using 32P- and 3H-labeled oligodeoxyribonucleotide probes.
Authors: Penschow J D , Haralambidis J , Coghlan J P
Published: April 2011

We investigated the location of expression of mouse glandular kallikrein genes in the submandibular gland of adult male mice at the ultrastructural level by hybridization histochemistry, using 32P- and 3H-labeled oligodeoxyribonucleotide probes. Vibratome slices were hybridized, flat-embedded, sectioned, and autoradiographs prepared. The 32P-labeled probe, which was specific for a region common to all mouse glandular kallikrein mRNAs, provided resolution at the cellular and subcellular level, demonstrating mRNA transcripts encoded by the majority of the 12 mouse glandular kallikrein genes in the perinuclear area of granular convoluted tubule cells (GCT) associated with rough endoplasmic reticulum (RER). The 3H-labeled probe was specific for mRNA transcripts of mGK-6, the renal kallikrein gene, which is also expressed in salivary glands. Occasional morphologically distinct granulated cells within GCTs, as well as striated duct cells, were found to express this gene. Resolution obtained with this 3H-labeled probe showed mGK-6 mRNA in striated duct cells to be located on RER and in the nucleus and perinuclear area of the cell. There was also an apparent mitochondrial association with regions of RER that labeled with this probe. The location of hybrids was confirmed by simultaneous assessment of sites of silver grains in serial sections. There are therefore at least two types of mGK-6-expressing cells in ducts of the submandibular gland, which are distinct from those expressing other kallikrein genes. In striated duct cells, there is evidence of a close mitochondrial association with RER that contains labeled mGK-6 transcripts, which is unexplained.
Pinocytosis as a select marker of ramified microglia in vivo and in vitro.
Authors: Ranson P A , Thomas W E
Published: April 2011

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.
Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease.
Authors: Siedlak S L , Cras P , Kawai M , Richey P , Perry G
Published: April 2011

Neurofibrillary tangles (NFT) are abnormal filamentous inclusions that develop in neurons in Alzheimer disease and other disorders. When neurons die, the neurofibrillary tangles that persist in the extracellular space show ultrastructural and antigenic changes. Both intra- and extracellular NFT have recently been shown to contain heparan sulfate proteoglycans (HSPGs). HSPGs are also present in other amyloid deposits in the brain and in systemic amyloidoses. Basic fibroblast growth factor (bFGF) is a heparin binding growth factor which is involved in angiogenesis and also has neurite promoting activity. We now report that bFGF binds avidly to extracellular NFT. Alz-50, a monoclonal antibody (MAb) to an abnormal form of tau and bFGF binding label mutually exclusive subpopulations of neurofibrillary tangles. bFGF binding is abolished by heparinase or heparitinase treatment and therefore is most likely based on the presence of HSPG. Binding of bFGF is a specific and sensitive morphological method to distinguish intra- from extracellular NFT. As intracellular NFT, which also contain HSPGs, are not labeled by bFGF binding, this finding also suggests that HSPGs are modified when the NFT become extracellular.
Androgen receptor expression in human tissues: an immunohistochemical study.
Authors: Ruizeveld de Winter J A , Trapman J , Vermey M , Mulder E , Zegers N D , van der Kwast T H
Published: April 2011

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.
Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody.
Authors: Schiechl H
Published: April 2011

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.
Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.
Authors: Cammer W , Downing M , Clarke W , Schenkman J B
Published: April 2011

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.
Presence of adenosine deaminase on the surface of mononuclear blood cells: immunochemical localization using light and electron microscopy.
Authors: Aran J M , Colomer D , Matutes E , Vives-Corrons J L , Franco R
Published: April 2011

Adenosine deaminase, which is essential for lymphoid differentiation and function, has previously been considered to be a cytosolic enzyme. In this report we demonstrate that it can be found associated with the plasma membrane of lymphocytes. By means of immunological techniques using both light and electron microscopy, adenosine deaminase was localized on the external side of the plasma membrane of normal lymphocytes and monocytes. Since the enzyme expression differed depending on the type of cell examined, new hypotheses about the mechanisms involving purine metabolism in immune dysfunctions or immunodeficiency syndromes may be considered.
Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes.
Authors: de Graaf A , van Bergen en Henegouwen P M , Meijne A M , van Driel R , Verkleij A J
Published: April 2011

We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.
Changes in cell polarity during mitosis in rat parotid acinar cells.
Authors: Tamaki H , Yamashina S
Published: April 2011

We studied the ultrastructure and cytochemistry of mitotic parotid acinar cells in vivo after induction of mitosis by isoproterenol injection. With entrance of the cells into the division cycle, the Golgi apparatus lost its characteristic stacked structure and internal polarity among the cisternae, appearing as fragments distributed throughout the cytoplasm. These fragments consisted of electron-lucent vesiculotubular structures and electron-dense 70-nm vesicles; neither component showed thiamine pyrophosphatase activity, a marker for trans cisternae of the Golgi apparatus, but the 70-nm vesicles showed a positive reaction for osmium impregnation, indicating retention of the cis nature. The rough endoplasmic reticulum was dilated and fragmented. Recovery of the structure of Golgi apparatus and rearrangement of rough endoplasmic reticulum occurred in daughter cells during telophase. These changes were the same as those observed after drug-induced inhibition of protein transport. The secretory granules were not dispersed but were divided into two groups with which centrioles were closely associated. Both groups migrated with the centrioles as far as the next interphase. The distribution of 5'-nucleotidase on the luminal plasma membrane showed no change during the process of division, thus demonstrating that surface polarity was maintained during mitosis. These changes in organelle structure and distribution may be due to the conversion of cell function from a secretory to a mitotic action.
Measurement of proliferation nuclear and membrane markers in tumor cells by flow cytometry.
Authors: Kute T E , Quadri Y
Published: April 2011

Nuclear and membrane markers that have been related to proliferative activity were measured by flow cytometry. The markers studied were transferrin receptor (TR), Ki-67 antigen, and epidermal growth factor receptor (EGFR). Two-color analysis for DNA via propidium iodide binding and for antigen expression via either a direct or indirect immunofluorescence assay was performed on three different cell lines and a solid human tumor model. The three cell lines tested were MCF-7 (breast), K-562 (leukemia), and A431 (a squamous cell). The solid tumor was obtained by subcutaneous injection of A431 cells into an athymic nude mouse. Our results demonstrate that TR are cell-cycle specific and can be readily measured in the cell lines. Ki-67 antigen is also cell-cycle specific in the cell lines tested, but the mean channel specific fluorescence uptake varies in the cell types. Finally, the EGFR was observed only in the A431 cell line, with most cells equally expressing this receptor. A bimodal distribution of EGFR was observed in A431 cells obtained from a solid tumor grown in an athymic nude mouse system. This suggests that cell line analysis may not always represent what might be observed under in vivo conditions. There are advantages to flow cytometry measurements of these factors which might be useful in predicting how patients should be treated and possibly the prognosis of cancer patients.
Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.
Authors: Kelly C D , Carter N D , de Boer P , Jeffery S , Moorman A F , Smith A
Published: April 2011

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.
Lectinocytochemical specificity in human eosinophils and neutrophils: a reexamination.
Authors: VanWinkle W B
Published: April 2011

As with secretory granules in other cell types, many of the protein components that make up the cytoplasmic granules of human leukocytes are glycoproteins. It is not unexpected, therefore, that lectins specific for various carbohydrate moieties can be localized in these granules following appropriate protocols for specimen preparation and thin-section labeling. In this study, isolated human eosinophils and neutrophils were prepared for lectin-gold electron microscopic localization by procedures that involve no exposure to aqueous fixatives, buffers, or solvents (rapid cryofixation and molecular distillation drying), thus removing the potential problem of constituent extraction or translocation during so-called "wet chemical" processing. In contrast to other reports, data presented illustrate the specific binding of soybean agglutinin (SBA) to eosinophil granule matrices (not the crystalline cores), as well as to a population of granules in neutrophils. A similar labeling pattern for wheat germ agglutinin (WGA) was also seen, confirming the presence of N-acetyl-D-galactosamine and N-acetyl-D-glucosamine residues in eosinophil and neutrophil granule matrices. These studies emphasize the need for carefully designed specimen preparation as well as subsequent thin-section labeling procedures in lectinocytochemical studies.
Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.
Authors: Uchiyama Y , Nakajima M , Watanabe T , Waguri S , Sato N , Yamamoto M , Hashizume Y , Kominami E
Published: April 2011

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.
Monoclonal antibodies to type V collagen for immunohistological examination of new tissue deposition associated with biomaterial implants.
Authors: Werkmeister J A , Ramshaw J A
Published: April 2011

We developed a panel of highly specific monoclonal antibodies (MAb) against dog Type V collagen. Each antibody showed differential reactivities towards Type V collagen from other species. All the antibodies were highly reactive in conventional ELISA, as well as with electroblots of collagen after polyacrylamide gel electrophoresis using non-denaturing conditions. The MAb were shown to be suitable for the immunohistological detection of Type V collagen in tissue sections, although this normally required pre-treatment of sections with 50 mM acetic acid. In particular, the antibodies were shown to be useful for examining samples of a collagen-based biomaterial, a vascular prosthesis, after explant from evaluation in an animal model. This showed that Type V collagen was most prominent in regions of new tissue formation within the neointima, close to the inner surface of the prosthesis. The broad spectrum of differential reactivities allows the antibodies to be used for a wide range of experimental models. These MAb therefore provide a novel approach for the evaluation of biomaterial performance, particularly for collagen-based implants.
Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues.
Authors: Tada T , Ohkubo I , Niwa M , Sasaki M , Tateyama H , Eimoto T
Published: April 2011

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.
Novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods.
Authors: Bronson D D , Bidmon H J , Tomaschko K H , Stumpf W E
Published: April 2011

Cross-linked and uncross-linked acrylamide polymers were used to alleviate technical difficulties in cryosectioning and autoradiographic processing of marine arthropods (Pycnogonum litorale), larval insect tissues (Sarcophaga bullata), and amphibian (Xenopus laevis) oocytes. Rapidly polymerized cross-linked acrylamide was used to prepare sections from an animal with a hard thick cuticle, P. litorale. Fragmentation and compression artifacts caused by tissue density differences were avoided and internal tissues were well preserved. Our results indicate that closely applied external support eliminates difficulties typically associated with arthropod cryosectioning. Pre-polymerized, uncross-linked acrylamide provided similar protection when used for sectioning larval tissue from S. bullata. Soft tissues under the cuticle were undamaged in tracheal and epidermal preparations. This polymer was also an excellent embedding material for soft tissues such as salivary glands, quickly penetrating tissue cavities and eliminating air bubbles. Uncross-linked acrylamide was used as an embedding and culturing medium for oocytes from X. laevis. The polymers were non-toxic and allowed the preparation of thin frozen sections containing as many as 50 large oocytes per section.
SPECIFICITY IN THE ALDEHYDE-COUPLING REACTIVITY OF NUCLEATED ERYTHROCYTES
Authors: LESSLER M. A.
Published: April 2011

The nucleated erythrocytes of Amphiuma, Necturus, Rana pipiens, Rana catesbeiana, Siredon, and Gallus were exposed to a series of aldehyde-coupling reactions. The degree of coupling was indicated by subsequent blocking of Feulgen coloration measured visually and photometrically. Characteristic variation from species to species of the inhibition of Feulgen coloration was observed. These differences are interpreted as qualitative differences in the reactivity of the DNA complexes in the species studied.
A HISTOCHEMICAL SPECIFIC THIONINE-SO2 REAGENT AND ITS USE IN A BI-COLOR METHOD FOR DEOXYRIBONUCLEIC ACID AND PERIODIC ACID SCHIFF POSITIVE SUBSTANCES
Authors: VAN DUIJN P.
Published: April 2011

A histochemical specific blue aldehyde reagent containing thionine and SO2, in a medium of tertiary butyl alcohol and water is described.

The reagent, as far as visual observation goes, reacts in a constant high intensity with aldehyde locations over a period of at least five weeks. In artificial light however the shade of the dye-aldehyde combinations shows some variation.

This unexplained phenomenon may point to a complicated reaction mechanism or may be related to the metachromatic qualities of the dyes.

For this reason the use of the reagent in quantitation experiments still requires further study though it is more constant in shade and reaches a higher intensity in shorter times than previously described mixtures of thionine and SO2.

When Feulgen hydrolyzed sections are stained with this reagent followed by the periodic acid Schiff procedure an image results of blue nuclei combined with the well known red extranuclear PAS pattern.

With the exception of glycogen the PAS positive polysaccharides withstand the preceding Feulgen hydrolysis. By visual observation no indication of significant overlapping of colors was found.
INFLUENCE OF HORMONES UPON THE DISTRIBUTION OF ALKALINE PHOSPHATASE IN MOUSE ADRENAL CORTEX
Authors: ALLEN JOHN M. , SLATER JUDITH J.
Published: April 2011

The effect of a variety of hormonal treatments upon the distribution of alkaline phosphatase in the adrenal cortex of BALB/c Jax male mice has been investigated. In the intact male mouse alkaline phosphatase is present in the reticularis, inner fasciculata and, to a lesser extent, in the outer fasciculata. Castration abolishes alkaline phosphatase from the reticularis and inner fasciculata; slight activity remains in the outer fasciculata. Testosterone propionate treatment restores the distribution of enzyme as seen in the normal male. Estradiol benzoate treatment in castrates results in the development of strong activity in the outer fasciculata. ACTH initiates strong alkaline phosphatase activity in the connective tissue and blood sinuses of all cortical zones and in the connective tissue of the medulla. FSH, LH, STH, thyroxine, progesterone, cortisone acetate and desoxycorticosterone acetate have no effect upon alkaline phosphatase in the castrate.
A HISTOCHEMICAL AND CHROMATOGRAPHIC STUDY OF NORMAL AND ATHEROMATOUS HUMAN ARTERIES
Authors: HENDERSON ANNE E.
Published: April 2011

Some of the chemical findings of previous workers, on the type and distribution of aortic lipids have been confirmed by histochemical methods and have been extended to include other arteries.

With increasing age the total lipid was found to increase in the apparently normal artery. Cholesterol and its esters appeared in the 20-29 decade in the intima of the aorta. Subsequent decades showed a deposition in the intima of other vessels and the media of the aorta. Phospholipid deposition followed a similar pattern.

The following steroids have been demonstrated by paper partition chromatography in both the normal and abnormal aorta, cholesterol, cholest-5-ene-3β:7β-diol, cholest-5-ene-3β:7α-diol, cholestane-3β:5α:6β-triol, and cholest-5-ene-3β:24 or 25 diol.
THE EFFECT OF MUCOLYTIC ENZYMES ON TISSUES STAINED WITH THE PERIODIC ACID-SCHIFF TECHNIQUE
Authors: EIDINGER D. , GHOSH A.
Published: April 2011

The effect of commercial preparations of pectinase and pectinesterase on the distribution and intensity of periodic acid-Schiff reactions was investigated in tissues fixed in acetone, Bouin, Zenker-formol and Carnoy.

The periodic acid-Schiff staining of collagenous fibers in connective tissue, the lens, seminal vesicle secretion, and keratin from skin regardless of fixation was intensified after incubation of sections of these tissues with the enzyme preparations at 37°C. in 0.2 M acetate buffer for 6-48 hours. The intensification of these structures, which appeared to be independent of the type of fixation, was shown to be due to the adsorption of a carbohydrate component of the enzyme preparation supplying the additional 1,2-glycol groups which reacted with periodic acid-Schiff.

After acetone fixation only, the periodic acid-Schiff reactive material was removed from mucus, coagulating gland and prostate secretions, and was decreased in cartilage matrix and the brush border of kidney. These effects were attributed to a heat labile "mucinase" of unknown nature present in commercial pectinase which is active at an acid pH. The loss of staining of sublingual gland secretion noted after Zenker-formol fixation may also be attributed to this enzyme.
THE INFLUENCE OF COLD, INANITION AND INSULIN SHOCK UPON THE HISTOCHEMISTRY OF THE ADRENAL MEDULLA OF THE MOUSE
Authors: ALLEN JOHN M.
Published: April 2011

The release of adrenaline from the adrenal medulla of the mouse under conditions of insulin shock and inanition and the release of noradrenaline under conditions of cold stress have been demonstrated. Associated with the release of both adrenaline and noradrenaline is the development of high levels of alkaline phosphatase activity in the secreting cells. These observations support the following conclusions: (a) the cells containing materials demonstrable by potassium iodate oxidation are true noradrenaline-secreting cells and do not represent secretory phases of a common cell type; (b) the association of increased alkaline phosphatase activity with release of adrenaline or noradrenaline is probably indicative of increased secretory activity.
HISTOCHEMICAL LOCALIZATION OF SPECIFIC OXIDATIVE ENZYMES. IV. SOLUBLE OXIDATION-REDUCTION DYES AS AIDS IN THE HISTOCHEMICAL LOCALIZATION OF OXIDATIVE ENZYMES WITH TETRAZOLIUM SALTS
Authors: FARBER EMMANUEL , LOUVIERE CONNIE D.
Published: April 2011

1. In tetrazolium stains for the succinic dehydrogenase system, diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase, the rate of staining can he greatly increased by adding any one of a number of soluble oxidation-reduction indicators to the incubation medium. The characteristic staining patterns are not altered.

2. This modification of tetrazolium staining interposes a dye as a carrier between the enzyme and the tetrazolium salt and makes it possible to stain for enzymes which do not react directly with tetrazolium.
HISTOCHEMICAL LOCALIZATION OF SPECIFIC OXIDATIVE ENZYMES. V. THE DISSOCIATION OF SUCCINIC DEHYDROGENASE FROM CARRIERS BY LIPASE AND THE SPECIFIC HISTOCHEMICAL LOCALIZATION OF THE DEHYDROGENASE WITH PHENAZINE METHOSULFATE AND TETRAZOLIUM SALTS
Authors: FARBER EMMANUEL , BUEDING ERNEST , LOUVIERE CONNIE D.
Published: April 2011

Incubation of frozen sections of rat kidney with lipase abolishes the staining reaction for the succinic dehydrogenase system with blue tetrazolium alone or blue tetrazolium plus methylene blue, pyocyanin, Janus green B, azure C or azure B bromide. In contrast, staining with phenazine methosulfate plus blue tetrazolium is unaffected by prior incubation with lipase. Qualitatively similar results were obtained with neotetrazolium.

Particulate preparations of rat liver and kidney, normally capable of reducing either methylene blue or phenazine methosulfate in the presence of succinate, lose the ability to reduce methylene blue after incubation with lipase. In contrast, the reduction of phenazine methosulfate is unaffected by this treatment.

The succinic dehydrogenase activity of frozen sections of Ascaris lumbricoides or of homogenates of Ascaris muscle, unlike rat kidney and liver, is unaffected by incubation with lipase and differs in this respect from purified preparations of Ascaris succinoxidase which are dissociated into succinic dehydrogenase and one or more carriers by treatment with lipase.

The results support the conclusion that the succinic dehydrogenase system contains carriers linked to the dehydrogenase. The carrier is essential for reduction of blue or neo-tetrazolium alone or in combination with several soluble oxidation-reduction dyes and is localized in tissue sections by these agents. The dehydrogenase can reduce phenazine methosulfate and this dye coupled to a tetrazolium salt can be used for the histochemical localization of succinic dehydrogenase in tissues.
TWO TYPES OF ACIDOPHILS IN THE ANTERIOR PITUITARY OF THE FROG AND THEIR RELATION TO THE PERIODIC ACID SCHIFF REACTION
Authors: ORTMAN ROBERT
Published: April 2011

Koneff's ('38) modification of Mallory-azan reveals the presence of azocarmine, orange G, aniline blue, and chromophobic cells in the pars distalis of the frog.

A strong periodic acid Schiff (PAS) reaction, apparently of carbohydrate-containing material, uniformly distributed throughout the cytoplasm, is found in the azocarmine cells and in the aniline blue cells. Certain of the orange G cells contained PAS-positive material confined to cytoplasmic vesicles. The PAS-positive material is probably not glycogen. It was not possible to distinguish between the various PAS-reactive sites with the cytochemical methods employed in this study.
Light microscopic morphometric analysis of peroxisomes by automatic image analysis: advantages of immunostaining over the alkaline DAB method.
Authors: Beier K
Published: April 2011

The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.
Localization of calcium changes in stimulated rat mast cells.
Authors: Horoyan M , Soler M , Benoliel A M , Fraterno M , Passerel M , Subra H , Martin J M , Bongrand P , Foa C
Published: April 2011

We studied intracellular free, bound, and sequestered calcium in rat mast cells after various stimulations. The use of a fluorescent probe combined with digitized imaging on individual living cells demonstrated transient increases of free Ca2+ in the micromolar range. The use of histochemical techniques (K pyroantimonate and anhydrous fixation), together with X-ray microanalysis, energy electron-loss spectroscopy, and electron spectroscopic imaging, revealed large amounts of stored calcium within the cells (in the millimolar range). Chelation experiments and stimulations enabled us to identify at least two pools of bound calcium which exhibited different dynamic behaviors. Stimulation in the presence of EGTA did not modify calcium from granules, granule membranes, and heterochromatin, whereas it decreased calcium from other cell compartments. Stimulation triggered variations in the amount of bound calcium but they did not parallel free calcium movements. Hence, whereas free calcium is implicated in exocytosis, bound calcium may be involved in altogether different cell functions.
Mosaic lectin and enzyme staining patterns in rat skeletal muscle.
Authors: Kirkeby S , Bøg-Hansen T C , Moe D
Published: April 2011

We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10- and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sections with the lectins ConA, GS-II, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-II, and HPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There was no correspondence in the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-II gave rise to distinct differences in the staining intensity between muscle fibers. In 10-day-old rats all fibers in the muscles were moderately stained with ConA, HPA, and jacalin, whereas a chessboard staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak affinity for GS-II and jacalin, as shown by dot-blotting. After electrophoresis and blotting to nitrocellulose membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles.
Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.
Authors: Adams J C
Published: April 2011

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.
Uptake of maternal immunoglobulins in the enterocytes of suckling piglets: improved detection with a streptavidin-biotin bridge gold technique.
Authors: Kömüves L G , Heath J P
Published: April 2011

In ungulates, intestinal absorption of maternal immunoglobulins from colostrum plays a vital role in the acquisition of passive immunity during early neonatal life. In the present study we used post-embedding colloidal gold labeling to examine the intracellular localization of IgG in the jejunal enterocytes of miniature piglets suckled for 2 hr. Quantitation of the immunolabeling revealed that the most sensitive technique for IgG detection was the streptavidin bridge-gold technique. In this method, the LR White-embedded sections were labeled sequentially with biotinylated anti-porcine IgG, streptavidin, and biotinylated BSA conjugated to 10-nm colloidal gold. With this approach, we found the following sequence of maternal IgG accumulation: passage of IgG from colostrum through the brush border; binding to the apical plasma membrane; uptake in noncoated pits and invaginations; transport in endocytotic vesicles; and accumulation in granules in the apical cytoplasm.
Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.
Authors: Dirks R W , Van Dorp A G , Van Minnen J , Fransen J A , Van der Ploeg M , Raap A K
Published: April 2011

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.
Light microscopic immunohistochemical analysis of the distribution of group II phospholipase A2 in human digestive organs.
Authors: Kiyohara H , Egami H , Shibata Y , Murata K , Ohshima S , Ogawa M
Published: April 2011

The light microscopic and immunohistochemical distribution of human Group II phospholipase A2 (M-PLA2) in digestive organs of both human fetus and adult, with a new monoclonal antibody (MAb) against M-PLA2, was investigated semiquantitatively. The immunoreactivity was distributed similarly in the adult and fetal epithelium of the esophagus, duodenum, and small intestine, and in the acinar, islet, and duct cells of the pancreas. The epithelium of adult gallbladder was immunoreactive. Paneth cells, especially the secretory apparatus, were strongly immunoreactive. Hepatic Küpffer cells and macrophages of the adult spleen were also immunoreactive. These results suggest that these cells contain secretory-type Group II PLA2, which may be involved in host defensive mechanisms, such as phagocytosis in human digestive organs. In the adult colon, the immunoreactivity was observed only in the ascending colon and was not found in the transverse, descending, sigmoid, or rectal colon. The immunoreactivity was not found in fetal colon. Similarly, immunoreactivity was found in hepatocytes and Küpffer cells of adult liver but not in fetal liver. By contrast, strong immunoreactivity was observed in the epithelium of the fetal stomach but not in adult stomach except in gastric neck cells. This suggests that the expression of M-PLA2 may be related to cell differentiation in particular organs.
Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.
Authors: Ridderstråle Y , Wistrand P J , Tashian R E
Published: April 2011

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.
Application of two-color immunofluorescence staining to demonstration of T-cells and HLA-DR-bearing cells in rheumatoid synovitis.
Authors: Itoh J , Kinjoh K , Ohyama A , Nose M , Kyogoku M
Published: April 2011

We studied the localization of T-cells and HLA-DR antigen-bearing (DR+) cells in rheumatoid synovitis by employing an improved two-color immunofluorescent staining (TCIF) technique. With this technique we have successfully identified DR+ activated T-cells in the inflammatory synovium. T-cells expressed HLA-DR antigen when they were in contact with DR+ antigen-presenting cells (APC). In addition, activated T-cells showed characteristic distribution within the synovium: they were found around high endothelial venules, within lymphoid follicles, and in hyperplastic synovial lining, suggesting their involvement in the development of rheumatoid synovial lesions via interaction with synovial DR+ APC lineage cells. These findings may contribute to better understanding of the role of activated T-cells in the histogenesis of rheumatoid synovitis, a typical chronic inflammatory lesion.