Ultrastructural localization of sulfated complex carbohydrates with a modified iron diamine procedure.

A thiocarbohydrazide-silver proteinate (TCH-SP) sequence was applied to thin sections of specimens that had been reacted with the high iron diamine (HID) method for ultrastructural localization of sulfated complex carbohydrates. The exposure to TCH-SP enhanced the electron opacity of HID-reactive sites and increased the sensitivity of the procedure. This held true for HID-reacted specimens whether or not they had been post-treated with osmium tetroxide. However in those not postosmicated after HID, the contrast and specificity appeared superior, as sites of osmiophilia were densified equally in specimens exposed to HID, and unexposed controls, by the final osmium tetroxide-TCH-SP sequence. Staining of immature granules of developing polymorphonuclear neutrophils by HID was intensified by the post-treatment with TCH-SP. In addition, granules of blood mononuclear leukocytes and heterophagosomes of peritoneal macrophages revealed HID affinity and hence content of sulfated mucosubstance that was not evident without the TCH-SP steps. Control procedures which entailed initial exposure of the specimen to FeCl3 or MgCl2 solutions and treatment of thin sections with TCH-SP failed to impart density to these sites.     

An ultrastructural study of histochemical staining of complex carbohydrates in the mouse posterior vitreous body

The vitreous body contains complex carbohydrates that can be demonstrated morphologically. Vitreous hyaluronic acid is very soluble but it can be precipitated by cetylpyridinium chloride (CPC) while being cross-linked by glutaraldehyde. Oligosaccharide chains of vitreous glycoproteins are fixed with glutaraldehyde alone. Mouse eyes were fixed with glutaraldehyde or glutaraldehyde and CPC and the complex carbohydrates of the posterior vitreous cortex were studied by electron microscopy. Cationic dyes were used in the fixative or for block-staining on most fixed tissue blocks to allow detailed observations of complex carbohydrates. Most blocks were postfixed with OsO4. The hyaluronic-acid domain on vitreous collagen fibrils sequentially contracted and expanded in size with various histochemical manipulations. Contraction of the domain of hyaluronic acid generally indicates an increased charge density. OsO4 contributes considerable charge density upon forming osmate esters, but tissue postfixed with OsO4 contained large globular forms of hyaluronic acid rather than the small globules observed in non-osmicated preparations. A model is proposed to explain the seemingly paradoxical findings by reference to suggested mechanisms of polysaccharide-ligand-OsO4 interactions.     

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Synopsis The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature précrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of -granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

The influence of fixation on staining of glycosaminoglycans in glial cells.

Cytoplasmic staining of glial cells by Alcian blue in 0.40 M magnesium chloride can be demonstrated in sections of brain tissue fixed in formol-acetic acid and in Bouin's solution. Phosphate-buffered aldehyde fixatives, whether at neutral or low pH, fail to preserve stainable material.     

Influence of plastic embedding media on staining and morphology of lipid bodies

Summary The following light microscopy staining techniques were applied to plastic embedded tissues: Toluidine Blue, Sudan III, Sudan Black and Nile Blue sulphate. All these procedures stain the lipid bodies; however the Sudan III appears to be the most suitable.The ultramicroscopic appearance of lipid bodies of tissues embedded in Epon, Araldite and Vestopal W is presented. The effects of dehydration as well as of different electron microscopic staining techniques is also investigated.This work was supported by Grants of Consiglio Nazionale delle Ricerche of Italy: no. 115/1151/0-1247, no. 115/0815/0-1365.The excellent technical assistance of Miss Hermina Spiele is gratefully acknowledge.     

The influence of fixation procedure, embedding medium and section thickness on morphometric data in thyroid gland.

In this study, the effects of fixation procedures, embedding medium and section thickness on stereological measurements of normal thyroid were analysed. The following conclusions were drawn: A) the use of a single section for the analysis of a lobe is sufficient if this section is located in the central part of the lobe. B) fixation and embedding with glutaraldehyde-Epon leads to a larger shrinkage than Bouin-paraplast, but the difference between the two procedures is not significant. C) osmium post-fixation reduces the shrinkage induced by glutaraldehyde and lowers the axial deformation produced by sectioning. D) Bouin's fixative and paraplast embedding induce considerable shrinkage of the interstitial tissue. The shrinkage obtained with glutaraldehyde-Epon is less. However, it is still not known whether this difference is due to the fixative, or to the embedding procedure or to both. E) only in glutaraldehyde and osmium-fixed material, embedded in Epon, can follicles and colloids be assumed to be spherical in shape without significant errors.     

A triple ultrastructural immunogold staining method. Application to the simultaneous demonstration of three hypophyseal hormones

The triple ultrastructural immunocytochemical labeling technique described here is based on the use of three different antisera raised in two different animal species: rabbit anti-corticotropin (R-ACTH), guinea pig anti-prolactin (GP-LTH) and rabbit anti-gonadotropin (R-LH beta). Staining is carried out on both sides (A and B) of the same tissue section. First, side A is incubated with a mixture of R-ACTH and GP-LTH and then with a mixture of the two corresponding species-specific immunoglobulins (IgG) adsorbed respectively to 5 and 20 nm gold particles: goat (G) anti-R IgG 5 + G anti-GP IgG 20; second, side B is incubated with R-LH beta, followed by species-specific secondary antibodies adsorbed in 10 nm gold particles; G anti-R IgG 10. With this technique, we demonstrated, on the same thin section, ACTH, LTH, and LH cells. The immunocytochemical procedure used has proved useful for simultaneous ultrastructural localization, on the same thin section, of three different antigenic sites. This technique, applied to other materials, could provide interesting information in several biological fields.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Summary The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens.

The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.     

Interactive influence of phosphorus and iron on nitrogen fixation by soybean

Adequate supplies of phosphorus (P) and iron (Fe) to legumes have been shown to be crucial in obtaining high nitrogen fixation rates and growth. These responses are anticipated as a result of the high requirement for P in energy transfer processes in the nodule and for Fe as a constituent of nitrogenase and leghemoglobin. However, little attention has been given to documenting the response of nitrogen fixation rates resulting from concentrations of P and Fe that actually exist in nodules. In particular, an open question is whether there is an interaction between nodule P and Fe concentrations that maximize nitrogen fixation activity. This study was designed to induce various concentrations of P and Fe in the nodule and to measure the resultant nitrogen accumulation and nitrogen fixation rates. Plant nitrogen accumulation was linearly correlated with both nodule P and Fe concentration, and with total plant nitrogen fixation rate as measured by acetylene reduction rate. Therefore, total nitrogen fixation rate was also correlated with nodule P and Fe concentrations, but a higher linear correlation was obtained for Fe as compared to P concentration. Surprisingly, nodule ureide concentration, which is generally assumed to be a positive index of nitrogen fixation rate, was negatively correlated with nodule P and Fe concentrations. These results indicated that higher concentrations of P and Fe in the nodules not only stimulated higher nitrogen fixation rates, but were associated with an enhanced ability to export ureides from the nodules. Since there was a linear response to both P and Fe over the range of nodule concentrations induced in these experiments, no evidence for optimum interactive concentrations of these two elements in the nodules was obtained.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens

Summary The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.     

Chromogranin A as a marker of neuroendocrine histogenesis of tumours: an immunoelectron microscopic study with considerations about the influence of fixation and embedding media on immunolabelling

The ultrastructural localization of chromogranin A (Chr A) was studied in eleven neoplasias of the diffuse neuroendocrine system (3 pancreatic islet-cell tumours, 1 medullary carcinoma of the thyroid, 1 large bowel and 1 small bowel carcinoid tumours, 2 carcinoid tumours of the lung, 1 adenoma of the parathyroid gland, 2 pheochromocytomas of the adrenal gland). On account of the great influence of the technical treatment of the samples on the immunolocalization of Chr A, the effect of the following variables was studied in a case of pheochromocytoma: fixation in glutaraldehyde versus paraformaldehyde, postfixation in osmium tetroxide versus omission, embedding in epoxy resin versus acrylic resin. The method of choice for the better preservation of the antigenic character of the tissue was found to be fixation in 4% paraformaldehyde, omission of osmium postfixation and embedding in LRWhite acrylic resin; by this procedure we were able to find Chr A in the neurosecretory granules of all the studied cases, using three commercially available antibodies directed against Chr A. These findings further confirm that Chr A is a reliable marker for the study of neuroendocrine neoplasias by electron microscopy.