bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.
Authors: Fernandez M C , Olmedilla A , Alche J D , Palomino P , Lahoz C , Rodriguez-Garcia M I
Published: April 2011

We investigated the immunolocalization of the olive major allergen Ole e I and Ole e I-like proteins in pollen from several Oleaceae species [olive (Olea europaea), ash (Fraxinus excelsior), privet (Ligustrum vulgaris), lilac (Syringa vulgare), and forsythia (Forsythia suspensa)]. Crossreactions among different pollens were found in enzyme immunoassays. For immunolocalization with light microscopy we used the silver enhancement technique with three monoclonal antibodies (1D8, 10H1, and 16G2) that recognize three different epitopes of the allergen Ole e I. Our findings show that the silver enhancement technique is very useful when several antibodies are to be used for rapid screening of different materials. MAb 10H1 gave the most precise results and was selected for further immunolocalization studies with transmission electron microscopy. The epitope recognized by this MAb was localized exclusively in the endoplasmic reticulum in olive pollen. In lilac, privet, and ash pollen, most of the reactivity was also seen in the endoplasmic reticulum; however, the 10H1 epitope was not detected in forsythia pollen.
Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.
Authors: Tagge E , Chandler J , Tang B L , Hong W , Willingham M C , Frankel A
Published: April 2011

DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL-containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.
Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.
Authors: Ishida-Yamamoto A , Eady R A , Watt F M , Roop D R , Hohl D , Iizuka H
Published: April 2011

The cornified cell envelope (CE) is an insoluble, highly resistant structure formed beneath the plasma membrane of differentiating keratinocytes. It consists of various crosslinked precursor proteins, including involucrin and loricrin. However, neither the normal assembly process of CE nor its alteration in skin disorders has been fully characterized. In this study we analyzed CE formation in normal skin and in lesional psoriatic skin by immunoelectron microscopy. In the superficial granular cells of normal epidermis, involucrin labeling was detected along the plasma membrane, whereas loricrin staining was mostly distributed diffusely within the cells, occasionally with some additional granular aggregates within the cytoplasm and nucleus. Loricrin was also present predominantly on the desmosomal attachment plaques, colocalizing with desmoglein. In the cornified cells, involucrin labeling was reduced, whereas loricrin labeling was retained and continuously decorated the CEs, with relative sparing of the desmosomal areas. In typical psoriatic epidermis, involucrin staining was very intense but loricrin labeling was markedly reduced. The involucrin-positive CEs were formed precociously and further maturation of CE did not occur. These results suggest that formation of CE occurs sequentially, initially involving involucrin deposition and subsequently involving loricrin incorporation. Psoriatic epidermis demonstrates a lack of proper CE maturation.
Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.
Authors: Timar J , Liu B , Bazaz R , Honn K V
Published: April 2011

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.
Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells.
Authors: Mialhe A , Cassanelli S , Louis J , Seigneurin D
Published: April 2011

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.
Prostaglandin F synthase is localized to contractile interstitial cells in bovine lung.
Authors: Fukui M , Fujimoto T , Watanabe K , Endo K , Kuno K
Published: April 2011

It was recently found that certain cells in the alveolar septum of the bovine lung are enriched in prostaglandin F (PGF) synthase. In this study we used immunohistochemical techniques at both light and electron microscopic levels to further characterize the PGF synthase-positive cells. By double immunofluorescence staining of bovine lung cryostat sections, the alveolar septal cells labeled by anti-PGF synthase antibody were also intensely labeled for cytoplasmic actin but not for alpha-smooth muscle actin. This labeling pattern suggests that the PGF synthase-positive cells in the septum are "contractile interstitial cells," which resemble conventional fibroblasts but characteristically contain prominent bundles of actin filaments. Immunogold electron microscopy of ultra-thin frozen sections of bovine lung showed that alveolar interstitial cells extending long cytoplasmic processes and closely associated with alveolar capillaries were intensely labeled for PGF synthase. Capillary endothelial cells, alveolar epithelial cells, and some fibroblastic cells were devoid of labeling. On the basis of these findings, we conclude that PGF synthase is specifically expressed in contractile interstitial cells within the alveolar septum. The protein may be a useful marker for contractile interstitial cells, whose physiological function and role in various pathological conditions have not been characterized in detail.
Evidence for a nonlysosomal origin of the acrosome.
Authors: Martínez-Menárguez J A , Geuze H J , Ballesta J
Published: April 2011

We studied the biogenesis of the acrosome in sperm cells in immunogold-labeled ultrathin cryosections of rat testis, using a variety of antibodies against endosomal/lysosomal marker protein and acrosin, the major secretory protein of sperm cells. As expected, acrosomes and proacrosomal vesicles in the trans-Golgi region contained abundant acrosin. Rat lysosomal membrane glycoprotein (lgp) 120 and mouse lysosome-associated membrane protein-1 were not detectable in the acrosomal membrane. Similarly, the late endosomal markers cation-dependent and -independent mannose 6-phosphate receptors were absent from the acrosome and proacrosomal vesicles. Therefore, acrosomes do not exhibit these endosomal/lysosomal features. Apart from (pro) acrosomal vesicles, both spermatocytes and spermatids contained classical lysosomes (positive for rat lgp 120, mouse lysosome-associated membrane protein-1, and cathepsin D) that were negative for acrosin. Quantitative analysis of the immunogold labeling showed that spermatocytes express more mannose 6-phosphate receptors and lgp 120 than spermatids, whereas the opposite situation existed for acrosin. These data indicate differential synthetic activity of lysosomal and acrosomal constituents in different states of sperm differentiation. Together, our observations argue against a lysosomal /endosomal origin of the acrosome.
Identification of F-reticulocytes by two-stage fluorescence image cytometry.
Authors: Osterhout M L , Ohene-Frempong K , Horiuchi K
Published: April 2011

Precise determination of reticulocytes (young red blood cells) containing fetal hemoglobin (Hb F), F-reticulocytes, is important for assessment of the efficacy of drugs such as hydroxyurea and butyrate in elevating the levels of Hb F in patients with sickle cell disease (SCD) and beta-thalassemia. We developed a reliable and easily applicable method for determining F-reticulocytes using fluorescence image cytometry. Reticulocytes were first identified by preparing a monolayer smear of blood stained by acridine orange. Images of reticulocytes and of all cells were obtained for a selected area on the smear. After removing the acridine orange, cells containing Hb F (F-cells) in the same area were then identified by immunofluorescence. Using images of F-cells, reticulocytes, and all cells for the same fields it was possible to identify F-reticulocytes. To assess the validity of our two-stage staining method, we compared our results with those obtained by traditional methods. There was significant correlation of our method with the conventional immunofluorescence staining method for F-cells (r2 = 0.99; slope = 0.99) and with the accepted brilliant cresyl blue method for reticulocytes (r2 = 0.97; slope = 0.96). Heretofore, the ability to determine F-reticulocyte levels has been limited to a small number of laboratories possessing special equipment and techniques. The method presented here should be of great interest to many basic science and clinical investigators involved in studies evaluating synthesis of Hb F.
Distribution of beta1 integrin during development of chick tarsometatarsal skin in vivo and in vitro.
Authors: Akimoto Y , Obinata A , Endo H , Hirando H
Published: April 2011

To determine the role of beta1 integrin in chick tarsometatarsal skin development, we examined the localization of the beta1 integrin immunohistochemically in vivo and in vitro by light and electron microscopy. Beta1 integrin was present over the entire cell surface of undifferentiated epidermis at early stages (Days 5, 9, 13). Marked changes in the localization of beta1 integrin occurred during epidermal keratinization and stratification, i.e., expression of beta1 integrin decreased in the superficial and intermediate cell layers from Day 13 to Day 17. After 17 days in vivo, when keratinization of the epidermis was completed, the distribution of beta1 integrin was confined to the basal layer of the epidermis in a pericellular distribution. During all stages examined, fibroblasts in the dermis were also stained. Immunoelectron microscopic study revealed that beta1 integrin was located on the plasma membrane of keratinocytes and dermal fibroblasts. The change in beta1 integrin localization that occurred in vivo could be reproduced in cultures of developing skin in which keratinization (differentiation) or mucous metaplasia (transdifferentiation) had been induced in vitro by hydrocortisone or retinol treatment, respectively. A monoclonal antibody against beta1 integrin caused striking changes in the epidermal keratinization process and in the basement membrane structure in vitro, i.e., inhibition of keratinization and detachment of the basement membrane from the basal surface of the epidermis. These results indicate that beta1 integrin plays an important role in cell-cell and cell-extracellular matrix interactions, which are important for epidermal development of the tarsometatarsal skin.
A method of quantitative autoradiography for the spatial localization of proteoglycan synthesis rates in cartilage.
Authors: Buschmann M D , Maurer A M , Berger E , Hunziker E B
Published: April 2011

Incorporation of [35S]-sulfate into cartilage tissue indicates the synthesis of aggrecan, the large aggregating proteoglycan (PG) that endows cartilage with resistance to compression. Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfate but does not provide information on the spatial location of synthesis within the tissue. Such spatially specific information is necessary to determine which cell populations respond to diffusible factors and to correlate local mechanical events (e.g., deformation, interstitial fluid stress) to cellular biosynthetic responses. The aim of this study was to develop and characterize a liquid emulsion autoradiography technique, including an automated grain counting procedure, to derive spatial profiles of aggrecan synthesis rates in cartilage. We chose mature 10-14-month-old bovine humeral head articular cartilage as a model system and applied a liquid emulsion autoradiography technique to [35S]-sulfate-labeled, resin-embedded, and semithin-sectioned tissue explants. High-magnification light microscopy color images were captured on a computer. Automated image analysis for grain number determination included a color thresholding procedure to discriminate grains from the lightly stained structural image and computation of the average area of a single grain from each image. Determination of grain number, whether originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a single grain in the same image. This procedure largely eliminated the effects of variations of microscope light intensity, camera performance, image focus, section stain intensity, and thresholding on the resulting grain numbers. By altering the specific activity of the medium radiolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive content in the underlying section. By cutting specimens in half and performing liquid scintillation counting on one half and autoradiography on the other half, we found that each disintegration occurring in the section during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58). Therefore, counted grain numbers can be directly converted to incorporated sulfate, largely reflecting the synthesis of the PG aggrecan. As an example of calculated intratissue profiles of aggrecan synthesis rates, we found for the mature bovine tissue in serum-free medium that aggrecan synthesis increases monotonically from the articular surface to the radial zone by as much as tenfold.
Differential distribution of S-100 protein and vimentin in the hypophyseal pars tuberalis of the guinea pig.
Authors: Kameda Y
Published: April 2011

In the hypophyseal pars tuberalis of guinea pigs, I examined immunohistochemical localization and development of vimentin and S-100 protein in comparison with those of the pars distalis. In the pars distalis, almost all folliculostellate cells expressed intense immunoreactivity for vimentin. A subpopulation of vimentin-immunoreactive folliculostellate cells was also immunoreactive for S-100 protein. During fetal development, vimentin-immunoreactive cells appeared at mid-gestation in the pars distalis and became numerous at late stages, whereas only a few S-100 immunoreactive cells were observed even at late stages. In the pars tuberalis, a large number of vimentin-immunoreactive cells were distributed in the cranial region surrounding the median eminence and the dorsocaudal region surrounding the infundibular stalk. These cells, however, were sparse in the ventrocaudal region in continuity with the pars distalis. Conversely, dense distribution of S-100 immunoreactive cells was restricted in the ventrocaudal region. Vimentin-immunoreactive cells were elongated and were mostly distributed as solitary cells, whereas S-100 immunoreactive cells were gathered in large or small cell groups and frequently formed colloid-containing follicles. During fetal development, large cell groups immunoreactive for S-100 protein were detected at late stages in the ventrocaudal region. Therefore, in the pars tuberalis S-100 immunoreactive cells are distinct from the vimentin cells and do not correspond to the folliculostellate cells of the pars distalis.
Ultrastructural immunogold localization of vimentin and S-100 protein in guinea pig pars tuberalis.
Authors: Kameda Y
Published: April 2011

Ultrastructural features of vimentin- and S-100 protein-positive cells in guinea pig pars tuberalis were examined by immunoelectron microscopy with the postembedding immunogold method. Interstitial cells that exhibited elongated cytoplasm and partly surrounded the specific secretory cells were filled with intermediate filament bundles on which immunogold particles for vimentin were densely located. Small colloid-containing follicles were occasionally distributed in the cranial region surrounding the median eminence. The follicular cells lining the luminal cavities contained considerable amounts of intermediate filaments immunoreactive for vimentin. Some specific secretory cells displayed various amounts of intermediate filaments immunoreactive for vimentin, which were scattered throughout the cytoplasm or concentrated around the nucleus. The specific secretory cells in which many parallel profiles of rough endoplasmic reticulum (RER) or a large number of mitochondria were dispersed throughout cytoplasm, i.e., being highly metabolically active, were devoid of intermediate filaments. The ventrocaudal region in continuity with the pars distalis was occupied by cells that were packed with vesicular inclusions, considered to be dilated cisternae of RER, and frequently formed colloid-containing follicles. Immunoreactivity for S-100 protein was located exclusively in this novel cell type. Immunogold particles for S-100 protein were distributed in the cytoplasmic matrix and were also associated with the membrane of vesicular inclusions. Gold particles were also detected on the tight junctions, desmosomes, and fibril materials at the apical cell regions of the colloid-containing follicles. The finding of two separate populations of cells containing vimentin and S-100 protein, respectively, supports the idea of functional separation in the pars tuberalis.
Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.
Authors: Monosov E Z , Wenzel T J , Lüers G H , Heyman J A , Subramani S
Published: April 2011

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.
Lamina propria macrophages in the human gastrointestinal mucosa: their distribution, immunohistological phenotype, and function.
Authors: Nagashima R , Maeda K , Imai Y , Takahashi T
Published: April 2011

In this study we systematically investigated the cellular distribution, immunohistochemical phenotype, and mucosal disposal function of macrophages in the lamina propria of the human gastrointestinal mucosa (lamina propria macrophages; LPMs). In all tissues examined, most of these LPMs accumulated beneath the epithelial layer that covered the apex of the lamina propria of the mucosa. These cells expressed normal levels of common macrophage markers such as CD68, LN5, lysozyme, ferritin, and alpha 1-anti-chymotrypsin. In addition, they expressed high levels of 25F9 (a market for a certain subpopulation of macrophages), MHC Class II molecules, and CD74 (MHC Class II-associated invariant chain). Interestingly, LPMs possessed some epithelial cell-associated antigens such as cytokeratin, carcinoembryonic antigen (CEA), and Ber-Ep4 in their cytoplasm. Ultrastructurally, these antigens were associated with cellular debris ingested by LPMs, which were recognized as apoptotic fragments by in situ end-labeling. Furthermore, double positive-labeled granules were seen in LPMs by double staining for epithelial cell-associated antigens and in situ end-labeling. These observations suggest that one of the major functions of LPMs is the disposal of apoptotic epithelial cells and that LPMs may be involved in the regulation of mucosal epithelial renewal.
Nonradioactive in situ hybridization for detection of mRNAs encoding for peroxisomal proteins: heterogeneous hepatic lobular distribution after treatment with a single dose of bezafibrate.
Authors: Schad A , Fahimi H D , Völkl A , Baumgart E
Published: April 2011

We present a nonradioactive in siru hybridization (ISH) protocol for detection of mRNAs in rat liver encoding for three peroxisomal proteins: catalase and urate oxidase as representatives of high-level abundance mRNAs and trifunctional protein (PH) as that of low-level abundance mRNAs. In addition to normal rats, animals treated for 24 hr with a single dose of bezafibrate were studied. The use of perfusion-fixation with 4% depolymerized paraformaldehyde/0.05% glutaraldehyde combined with paraffin embedding and the application of digoxigenin-labeled cRNA probes provided optimal cytological resolution and high sensitivity comparable to that of radioactive ISH. In parallel experiments, the same digoxigenin-labeled cRNA probes were used for Northern and semiquantitative dot-blot analysis of isolated RNAs. In control animals, the mRNAs for catalase and urate oxidase were uniformly distributed across the liver lobule and were confined to liver parenchymal cells. The bile duct epithelial and the sinusoidal cells remained negative. The specificity and the high resolution of our protocol were further substantiated by reciprocal localization of transcripts for albumin and glyceraldehyde-3-phosphate dehydrogenase in different regions of the liver lobule and for catalase in the proximal tubules of the renal cortex. Whereas in control livers the transcripts for PH were barely detectable, a strong signal was found in pericentral hepatocytes of bezafibratetreated animals, corresponding to an 8-10-fold increase of mRNA detected in dot-blots. In contrast, the urate oxidase mRNA was reduced by more than 50%, with diminution of staining in pericentral regions of the liver lobule. The mRNA encoding for catalase was only slightly affected. Further applications of this protocol should be helpful in elucidation of the cell-specific transcriptional regulation of peroxisomal proteins in various organs under normal and pathological conditions.
Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils.
Authors: Taipale J , Saharinen J , Hedman K , Keski-Oja J
Published: April 2011

We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.
Estimation of the C-cell numbers in rat thyroid glands using the optical fractionator.
Authors: Feinstein R E , Westergren E , Bucht E , Sjöberg H E , Grimelius L
Published: April 2011

We estimated the total number of calcitonin-immunoreactive C-cells in rat thyroid gland using the optical fractionator, the unbiased stereological method for estimation of numbers. It was necessary first to use a fixative composed of formalin, acetic acid, and ethanol to distinctly visualize the C-cells. The 40-microm-thick sections had to adhere to chromalum-gelatin-coated Superfrost Plus glass slides, and the immunostaining technique had to stain the C-cells evenly throughout the whole sections. Because the C-cells were irregularly distributed in the thyroid tissues, their counting required screening of about 500 fields per lobe, but the number of C-cells counted need not be high, about 90 per lobe. We estimated that rats have 185,000 +/- 42,000 C-cells (mean +/- SD; n - 7). The C-cell population did not differ significantly between the two lobes of a given rat, but it varied markedly among rats. The biological differences among the animals contributed 83% to the observed variability, whereas the methodological uncertainty contributed 17%. The serum levels of calcitonin and calcium were not closely correlated to the C-cell numbers. Our results indicate that variability in C-cell experiments can be reduced most effectively by increasing the number of animals used. However, the similar C-cell frequency found in the two thyroid lobes of each rat allows the use of one uniformly sampled lobe for quantification and the other lobe for further analysis.
Multi-organ reactivity of a monoclonal natural autoantibody that promotes remyelination in a mouse model of multiple sclerosis.
Authors: Miller D J , Njenga M K , Parisi J E , Rodriguez M
Published: April 2011

A contemporary view of autoimmunity suggests that self-reactivity is a normal phenomenon, in contrast to the classical association between autoimmunity and immunopathology. We have previously demonstrated that monoclonal antibody SCH94.03, a natural autoantibody with polyreactivity towards several purified protein and hapten antigens, promotes central nervous system remyelination when passively transferred to SJL/J mice chronically infected with Theiler's murine encephalomyelitis virus, an established experimental model of multiple sclerosis. In this study we characterized the autoreactivity of SCH94.03 with endogenous mouse tissue using immunoperoxide and multiple-color immunofluorescence staining techniques on frozen tissue sections. Within the nervous system, SCH94.03 labeled fibrous astrocytes, ependymal cells, ganglion satellite cells, and a sub-population of microglia, oligodendrocytes, and peripheral nervous system neurons. Outside the nervous system, SCH94.03 labeled gastrointestinal tract smooth muscle and luminal epithelium, erythrocytes, and interdigitating dendritic cells in peripheral lymphoid organs. These data indicate that SCH94.03 is a multi-organ reactive autoantibody and support the hypothesis that autoantibodies can have a beneficial rather than a pathogenic function in central nervous system demyelinating diseases.
OBSERVATIONS ON NAPHTHOL STAINING AND THE HISTOCHEMICAL LOCALIZATION OF ENZYMES BY NAPHTHOL-AZO DYE TECHNIQUE
Authors: DEFENDI V.
Published: April 2011

A group of naphthols—1- and 2-naphthol, 6-benzoyl-2-naphthol and 6-bromo-2-naphthol—have been investigated in relation to their tissue affinity. It has been found that naphthol binding is mainly dependent on the solubility of the naphthols and on the type of tissue considered. Time of incubation, naphthol concentration and pH also play in important role in the intensity of the resulting stain. 1- and 2-naphthol have very little affinity for cell constituents, while 6-bromo-2-naphthol and 6-benzoyl-2-naphthol are strongly bound by cellular structures. The affinity of the latter two naphthols is specific for cell parts and tissue type: the cytoplasm of epithelial cells is strongly stained, while connective tissue, kidney glomeruli and inner medulla and cell nuclei at the concentration used devoid of any naphthol binding.

Correlated experiments have been carried on the histochemical demonstration of enzymes utilizing the esters of these naphthols, by simultaneous and post-incubation coupling methods. Considerations have been offered on the importance of specific tissue and cellular affinity of insoluble naphthols in relation to the reliability of histochemical localization of enzymes by the post-incubation coupling technique.
CYTOCHEMICAL STUIDIES OF KERATIN FORMATION AND OF EPITHELIAL METAPLASIA IN THE RODENT VAGINA AND PROSTATE
Authors: BERN HOWARD A. , ALFERT MAN , BLAIR SIDNEY M.
Published: April 2011

Quantitative and qualitative cytochemical studies were made of keratinized epithelia from the estrogen-stimulated vagina and metaplastic prostatic lesions of the mouse, and from the estrogen-stimulated and avitaminotic-A vagina of the rat.

Karyometric and general cytochemical observations indicate a certain minimal testoid stimulation of the anterior prostate in the castrate mouse.

Nuclear changes during keratinization represent a degenerative process different from that seen in pycnosis. In keratinizing epithelia the nuclei first swell and then shrink, and both of these changes are accompanied by loss of DNA and protein from the nuclei.

Metaplastic epithelium in prostatic lesions developing as a result of estrogen treatment can be distinguished from the original epithelium not only by its alkaline phosphatase activity, but also by its increased cytoplasmic RNA and protein accumulations.

Definite histologic and cytochemical differences exist in the rat between the keratinized vagina seen in vitamin A deficiency and that seen after estrogen administration. In addition to such features as the presence of longitudinal epithelial folds and of leukocytic infiltration in the former, the estrogen-stimulated epithelium shows intense alkaline phosphatase activity and distinctive distribution of RNA, protein, and protein-bound SH groups. These differences are possibly explicable on the basis of a greater rate of proliferation of the estrogen-treated epithelium.
A TRYPTOPHAN HISTOCHEMICAL METHOD
Authors: BRUEMMER NANCY C. , CARVER MICHAEL J. , THOMAS LLOYD E.
Published: April 2011

1. A new tryptophan histochemical procedure is described.

2. The results of tests on the specificity of the reaction are reported.

3. The results of measurements of the infra-red absorption of the colored reaction product (using free tryptophan) and other similar compounds are reported and a possible structure of the reaction product is proposed.
THE INFLUENCE OF ALLOXAN AND DIETARY RESTRICTION ON RAT LIVER DESOXYRIBONUCLEIC ACID (DNA)
Authors: CONRAD EUGENE A. , BASS ALLAN D.
Published: April 2011

Rats fasted for 48 hours showed a decrease in liver mass and total number of nuclei. Although the liver content of DNA remains unchanged, an increase was noted in the amount of DNA per average nucleus, associated with an increase in the percentage of nuclei of higher ploidy.

Alloxan treatment results in an elevation of 10-20 per cent in the DNA content per average nucleus when compared to ad libitum fed control rats. A comparison of alloxan treated rats with pair fed control animals showed no significant difference when the following were compared: liver and body weights, total number of nuclei and DNA content per liver, the average amount of DNA per nucleus, and ploidy distributions. These data suggest that the effect of alloxan on rat liver nuclei is the result of associated dietary factors rather than an effect of the chemical on the liver nucleoprotein.
DIRECT OBSERVATION OF CELLULAR ABSORPTION BY ULTRAVIOLET TELEVISION MICROSCOPY
Authors: WILLIAMS GEORGE Z.
Published: April 2011

An instrument chain utilizing an ultraviolet light source, ultraviolet sensitive vidicon, camera, amplifier, monitor screen, and oscilloscope, facilitates the direct and rapid observation of the absorption properties of intact as well as fixed cells. The ultraviolet television microscope provides a means for rapidly scanning hundreds of cells and obtaining satisfactory comparative oscilloscopic tracings which express the qualitative absorption properties sufficiently for many biological purposes and is being improved to yield more accurate quantitative data. It may be used for accurate and quick focusing with ultraviolet light and camera. Living cells being scanned in a warm stage chamber may be moved through the beam so rapidly that exposure and injury are minimum. This instrument is most useful for directly observing cellular metabolic processes.
THE HISTOCHEMICAL DEMONSTRATION OF INDOLE DERIVATIVES BY THE POST-COUPLED P-DIMETHYLAMINOBENZYLIDENE REACTION
Authors: GLENNER G. G. , LILLIE R. D.
Published: April 2011

1. A highly specific, chromogenic, localizable and permanent histochemical method for the demonstration of indole derivatives by use of a post-coupled benzylidene reaction is described.

2. The mechanism of the reaction as well as its tissue specificity is discussed.

3. The localization of indole derivatives to such sites as the β-cells of the pituitary, the granules of blood and tissue eosinophiles, the granules of the Paneth cells in the small intestine, chief cell granules of the gastric fundus, zymogen granules of the pancreatic acinar cells, α-cells of the islets of Langerhans, fibrin,

[See figures in the PDF file]

fibers and cement substance of the lens, neurokeratin and thyroid colloid has been demonstrated.

4. The relationship between the known tryptophan content of proteins and the sites of a strongly positive benzylidene reaction is discussed as well as a similar correlation with sites of negative reaction.

5. There is a tryptamine-formaldehyde condensation product, that is not a tetrahydronorharman, and is benzylidene reactive.

6. The significance of the lack of staining of melanin and enterochromaffin substance by the post-coupled benzylidine reaction is discussed.
HISTOCHEMICAL STUDIES OF SUCCINIC DEHYDROGENASE OF THE BRAIN OF MICE, RATS, GUINEA PIGS AND RABBITS
Authors: SHIMIZU NOBUO , MORIKAWA NAGAAKI
Published: April 2011

Succinic dehydrogenase activity of the brain was histochemically investigated in mice, rats, guinea pigs and rabbits. Fresh frozen sections cut at 20 µ were stained with the Seligman-Rutenburg method as modified by Rosa and Velardo.

The pattern of enzyme distribution is remarkably similar but the intensity of staining differs somewhat in the animal species, being highest in mice and rats, intermediate in rabbits and lowest in guinea pigs. The enzyme activity is found exclusively in the grey matter and ocurs both in the perikaryon and neuropil. The activity in the grey matter exhibits the characteristic local variations which are ascribed to the varying participation of the nerve cell bodies and surrounding neuropils in the reaction. Nerve cell bodies contain variable amounts of dye particles of different sizes, and the neuropil possesses variable numbers of coarse granules. An intense reaction is given in the caudate nucleus, putamen, anterior nucleus of the thalamus, interpeduncular nucleus and cerebellar cortex. An intermediate activity occurs in the neocortex, rhinencephalon, optic tectum, most of the cranial nerve nuclei, superior and inferior olivary nuclei, and the Goll and Burdach nuclei. A low activity is present in the paraventricular structures, such as supraoptic crest, subfornical and subcommissural bodies, and area postrema, as well as the paraventricular and supraoptic nuclei of the hypothalamus, and dorsal nuclei of the vagus nerve.
STUDIES ON TISSUE FIXATION: THE PENETRATION OF TRICHLORACETIC ACID SOLUTIONS INTO RAT TISSUES
Authors: CAFRUNY E. J.
Published: April 2011

A method for measuring the rate of penetration of trichloracetic acid solutions into tissues was developed and the effects of a surface active agent, temperature, and concentration of fixative on penetration rates were evaluated.

Penetration distances into rat liver, kidney, and cerebral cortex were determined and expressed as a function of time.

It was found the penetration distances vary directly with temperature and fixative concentration.

Tergitol, a wetting agent, did not increase penetration rates into kidney or liver tissue and in high concentration actually decreased penetration into kidney.

Penetration distances were found to be proportional to the square root of the time of exposure of liver to 10% trichloracetic acid solutions but this proportionality did not hold for kidney and cerebral cortex.
ULTRACENTRIFUGAL STUDIES ON THE CYTOPLASMIC COMPONENTS
Authors: TAKANAMI MITURU
Published: April 2011

Ultracentrifugal analysis, electron microscopical observation and chemical analysis on the cytoplasmic homogenate of rabbit liver were performed in order to determine the constitution and diversity of the cytoplasmic components.

The result of experiments showed that the cytoplasmic components comprised 1) a heterogeneous granular component, sedimented at a rate more than 250s, 2) two closely spaced components whose sedimentation rates were 51s and 64s respectively and 3) three smaller components with rates of 4s, 6s and 12s with regard to the sedimentation analysis. The first heterogeneous granular component showed continuous distribution of size and shape from 50-60 mµ to several µ in diameter and contained three chemical materials, ribonucleic acid, lipids and protein components. The next two closely spaced components, corresponding with considerable numbers of homogeneous micro-particles of about 20 mµ electron microscopically, represented a ribonucleoprotein component in which the RNA:protein ratio was about 1:1.7 and a metalloprotein containing about 20% iron respectively. Accordingly, I must emphasize the need to discriminate these macromolecular components from the heterogenous granular component, including the so-called large granule and small granule fractions, for biochemical Studies on the cell components, and that the cell-fractionation techniques should be improved according to present ultracentrifugal data.
INTRACELLULAR LOCALISATION OF DEHYDROGENASE SYSTEMS USING MONOTETRAZOLIUM SALTS AND METAL CHELATION OF THEIR FORMAZANS
Authors: PEARSE A. G. E.
Published: April 2011

1) A method is proposed for the demonstrations of dehydrogenases and diaphorases using the principle of simultaneous chelation of an enzymically produced monoformazan by means of metallic ions.

2) Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium and M/60 cobalt a black metal-formazan complex is produced. Its nature is such that fine intracellular localisation becomes possible.

3) Chelation occurs under histochemical conditions with cobalt, copper, nickel and uranium and it is suggested that the electron opacity so induced may facilitate localisations of dehydrogenases on the electron microscope level.
TETRAZOLIUM REDUCTION AS A MEASURE OF MONOAMINE OXIDASE ACTIVITY IN VITRO
Authors: WEISSBACH HERBERT , REDFIELD BETTY G. , GLENNER GEORGE G. , MITOMA CHOZO
Published: April 2011

1 . Data has been presented showing that tetrazolium salts can be used for the histochemical localization of monoamine oxidase, although there are limitations to its application to in vitro studies. These limitations have been discussed.

2. It appears that an indole carbonyl compound is necessary for 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction. A diaphorasetype system may be required to transfer the electrons from the indole carbonyl intermediate to INT.
REACTION OF TISSUES WITH SCHIFF'S REAGENT AFTER TREATMENT WITH ANHYDROUS CHROMYL CHLORIDE
Authors: MENDE THOMAS J. , CHAMBERS EDWARD L.
Published: April 2011

Oxidation with chromyl chloride in an anhydrous solvent followed by exposure to Schiff's reagent can be used for the demonstration of glycogen and mucins in tissue sections. The intensity of the reaction is weaker than in the periodic acid-Schiff technique and other substances besides glycogen and mucins generally fail to react. Acetylation and deacetylation abolish and restore the reaction respectively.

On prolonged exposure to chromyl chloride at room temperature a typical Feulgen nucleal reaction is obtained. Treatment with the following inorganic acid chlorides in carbon tetrachloride also results in Feulgen staining: sulfuryl chloride thionyl chloride and phosphoryl chloride. Current views concerning the mechanism of aldehyde group liberation in the Feulgen reaction are discussed in connection with these results.
A MODIFICATION OF THE KOELLE TECHNIQUE FOR USE WITH OXIMES
Authors: BERGNER A. DOROTHY , O'NEILL JOHN J.
Published: April 2011

The Koelle histochemical method has been modified to study the effect of oximes on cholinesterase activity at motor endplates. It is possible by lowering the pH to hold the reaction between substrate and oxime to a minimum. With this modified procedure, the inhibitory effect of pyridine-2-aldoxime methiodide and diacetylmonoxime on motor endplate cholinesterase can be demonstrated.
IMPROVEMENT IN THE HISTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE BY USE OF NITRONEOTETRAZOLIUM CHLORIDE
Authors: PEARSON BJARNE
Published: April 2011

A new method for the histochemical localization of succinic dehydrogenase activity has been described in which the resultant formazan is free of crystal formation.

The resultant formazan is extremely fine and granular permitting accurate and precise localization of the enzyme.

The tetrazolium salt used was 2,2'-diphenyl-5,5' - di - ( m - nitrophenyl) - 3,3' - (4,4' - biphenylene) ditetrazolium chloride.

Minimal granular artifacts which appear may be eliminated by dissolving the tetrazolium salt in N,N-dimethylformamide and rinsing the slide in 80% alcohol following incubation. This has a tendency to dissolve fat and prevent aggregation of granular artifacts which may appear subsequently upon storage of slides.
HISTOCHEMICAL DEMONSTRATION OF β-D-GALACTOSIDASE IN THE RAT
Authors: RUTENBURG ALEXANDER M. , RUTENBURG SELMA H. , MONIS BENITO , TEAGUE RUTH , SELIGMAN ARNOLD M.
Published: April 2011

A histochemical technic for demonstration of β-d-galactosidase in formalin fixed sections has been presented. Enzymatic activity was best demonstrated in frozen sections of the kidney, liver, gastrointestinal tract, testis, epididymis and vas deferens of the rat and to a lesser extent in salivary gland, pancreas, thyroid, trachea, lung, spleen and lymphoid tissue. Leucocytes stained intensely. Enzymatic activity was largely present in the cytoplasm of epithelial cells. Cell nuclei, myocardium, connective tissue and erythrocytes were inactive.
THE NILE BLUE REACTION OF PEPTIC GLAND ZYMOGEN GRANULES: THE EFFECT OF METHYLATION AND ALKALI DEMETHYLATION
Authors: LILLIE R. D.
Published: April 2011

The zymogen granules of the chief cells of the peptic glands are colored dark blue when the sulfuric acid Nile blue technic for the demonstration of lipofuscin is applied. Weaker blue colors appear in smooth muscle, soft keratin and certain cytoplasms. After methylation the same technic colors these structures in varying shades of pink to red.

Decomposition of the methyl esters by KOH restores the blue staining. Extraction with acetone directly after staining with sulfuric acid Nile blue prevents both the blue and the pink to red colorations. The strong Nile blue staining of peptic gland zymogen granules may correlate with the large excess of free anionic residues over cationic in pepsin, and the considerable excess of aspartic and glutamic acids over arginine, histidine and lysines in the same protein.

Use of alcoholic potassium hydroxide affords a convenient histochemical procedure for conversion of esters to fatty acids, provided the acids are not soluble in the alkali.
A NEW CARBOWAX METHOD FOR ROUTINELY PERFORMING LIPID, HEMATOXYLIN AND EOSIN AND ELASTIC STAINING TECHNIQUES ON ADJACENT FREEZE-DRIED OR FORMALIN-FIXED SECTIONS
Authors: ZUGIBE FREDERICK T. , KOPACZYK KRYSTYNA C. , CAPE WILLIAM E. , LAST JULES H.
Published: April 2011

1. A freeze-drying method is presented for the simultaneous processing of a large number of tissue specimens.

2. An infiltration medium consisting of carbowax "1000" and a blocking mixture consisting of carbowax "1000" and carbowax "4000" were developed and resulted in ribbons which could be cut as fine as 2 µ.

3. Diffusion currents encountered during flotation were obviated by the use of a General Flotation Solution consisting of potassium dichromate, glycerin and gelatin for routine sections and a Special Flotation Solution consisting of carbowax and glycerin for certain fragile tissues.

4. The application of routine stains to adjacent carbowax sections included a hematoxylin and cosin technique with good cosin differentiation and sharp hematoxylin staining, an elastic-collagen technique demonstrating distinct elastic and collagen fibers and a lipid technique with minimal fat loss.

5. This carbowax method allows for correlative, multiple staining and ultramicrochemical analysis on adjacent tissue sections.