Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.     

The adrenal gland of Triturus carnifex after glucagon administration

The present work was undertaken in order to investigate the influence of endocrine pancreas on the adrenal gland of Triturus carnifex. Our experiments aimed at studying the effects of intraperitoneal injections of glucagon on ultrastructural morphological and morphometrical features of steroidogenic and chromaffin tissues, as well as serum levels of aldosterone, corticosterone, norepinephrine (NE) and epinephrine (E). With regard to steroidogenic tissue, in January and November, glucagon decreased lipid droplet content in steroidogenic cells, that showed clear signs of increased activity. Moreover, increased corticosteroid serum levels were found. With regard to chromaffin tissue, in January glucagon played a stimulatory role on PNMT enzyme, eliciting an increase in the presence of E granules, and a decrease in the presence of NE granules, in the chromaffin cells. Moreover, increased E serum levels and decreased NE serum levels were found. In November, glucagon gave rise to a decrease in the presence of NE and E granules in the cells; E serum levels were strongly increased, whereas NE serum levels did not undergo any significant change. These findings suggest an involvement of the endocrine pancreas of the newt in the modulation of adrenal gland activity.     

Localization and characterization of carbohydrates in adrenal medullary cells

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.     

Recognition of cells in mitosis by flow cytofluormetry.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.     

Bismuth Hematoxylin Stain for Arginine Residues

Bismuth ions complex with hematoxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Extraction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of arginine completely abolishes staining.     

Stains for A, B, and D cells in fetal rat islets.

Ten techniques often used for identification of A, B, and D cells in adult islets of Langerhans were applied to fetal rat pancreas. Modifications were tried with many of these techniques. Two indole methods (xanthydrol and postocoupled benxylidene reactions) and a cryostat technique using o-phthaladehyde failed to stain fetal islets. Phosphotungstic acid hematoxylin and lead hematoxylin lightly stained fetal A cell granules in Helly's fixed tissue. The Grimelius silver nitrate technique stains adult rat A cells but failed to stain fetal cells. A modification of this technique stained fetal A cells and a possible 4th cell type. The specificity of this method was confirmed by restaining stained cells with a fluorescent antibody technique and with pseudoisocyanin. B cells, as previously reported, were readily stained by the aldehyde fuchsin technique. Fetal D cells were not stained by the Hellerstrom-Hellman alcoholic silver nitrate method, nor did they display pseudoisocyanin metachromasia after acid hydrolysis; they did fluoresce brightly with this technique when viewed with UV light. It was thus possible to distinguish the three usual cell types, plus a possible fourth type, in the fetal rat pancreas.     

Purification and crystallization of recombinant Escherichia coli malate dehydrogenase

Malate dehydrogenase from Escherichia coli has been crystallized with polyethylene glycol and citrate buffer at pH 5.7. The enzyme was obtained from an E. coli strain in which the chromosomal malate dehydrogenase gene was contained on a pBR322 vector. Two types of crystals have been observed; a monoclinic C2 form and an orthorhombic C222(1) form, which is found infrequently. Monoclinic crystals were used as seeds in several rounds of crystallization until large crystals suitable for diffraction analysis were available. A complete X-ray data set to 2.0 A has been collected.     

Application of ultrafiltration method to measurement of catecholamines in plasma of human and rodents by high-performance liquid chromatography

In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000 x g at 4 degrees C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20-400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.     

Adrenal medullary basophilia in ox,pig and sheep

Summary In ox, pig and sheep the adrenaline storing cells are intensely basophilic compared with the noradrenaline storing cells when aldehyde fixed tissue is stained with toluidine blue at pH 5.0 and above. This has been shown to be due to carboxyl groups from the glutamate rich chromaffin granule soluble protein. In isolated chromaffin granules adenosine nucleotides also bind the dye. Fixation of adrenal medulla in agents not containing aldehydes, or the use of cryostat sections results in equal basophilia in the adrenaline and noradrenaline storing cells. The probable mechanism of the differential basophilia of the two sorts of medullary cells following aldehyde fixation is discussed.     

Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-life of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.     

Staining Mitochondria in Saccharomyces cerevisiae

After testing various procedures (amidoblack 10B, acid fuchsin-methyl blue, Luxol fast blue MBS-phloxine, toluidine blue O, Jams green B and pinacyanol), three stains can be recommended for staining both types of mitochondria (globose and threadlike) in the cells of Saccharomyces cerevisiae: (1) 0.1% solution of amidoblack 10B in citrate buffer (pH 3.0) for 10 min; (2) 0.01% solution of toluidine blue O in phosphate buffer (pH 6.0) for 30 min; (3) 0.01% solution of Janus green B in distilled water (pH 5.6) for 30 min. The latter stain is most specific because its staining reaction depends upon the action of the mitochondrial enzyme cytochrome c oxidase. Yet, low concentrations and short incubation periods must be applied to avoid poisoning of the cell metabolism.     

A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies

A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565-574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6), 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein (n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 +/- 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.     

Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.     

Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627

Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein(-1) extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 degrees C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 degrees C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.     

Peptide and amine producing endocrine-like cells in the chicken thymus

Summary The thymus of the chicken contains at least two types of endocrine-like cells predominating in the juxtacortical medulla. One type stores 5-hydroxytryptamine and is stained by the argentaffin, chromaffin and Schmorl methods. Treatment with reserpine markedly reduces its 5-hydroxytryptamine content. The other cell type is devoid of 5-hydroxytryptamine; if supplied withl-dopa it can produce and store dopamine. Both cell types stain with the argyrophil method of Grimelius and with HCl-basic dye methods believed to reflect the presence of peptides with masked carboxyl groups. In the electron microscope both cell types were found to contain numerous cytoplasmic 2000–3000 ? granules similar to those seen in polypeptide hormone-producing cells elsewhere. The cytoplasmic granules in one of the two endocrine-like cell types are argentaffin and chromaffin, indicating that they are the storage site for 5-hydroxytryptamine. It is suggested that the main secretory products of the two endocrine-like cell types are peptides, possibly regulating lymphatic tissue function.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.