Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

Optimization of immunogold labeling TEM. An ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen.

We developed an ELISA-based method for rapid optimization of various tissue processing parameters in immunogold labeling for electron microscopy. The effects of aldehyde fixation, tannic acid, postfixation, dehydration, temperature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the results confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) of glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some distortion of cellular ultrastructure but better antibody binding activity compared to dehydration up to 100%. Postfixation or incorporation of tannic acid in the primary fixative caused almost total loss of activity, whereas antigen retrieval of osmium-postfixed material resulted in approximately 90-100% recovery. The sensitivity of detection of proteins by immunogold labeling electron microscopy depends on the retention of antibody binding activity during tissue processing steps, e.g., fixation and dehydration. Our study indicated that an ELISA-based screening method of various tissue processing procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM.     

Coated microvesicles in central nervous system myelinated axons

The distribution of coated and uncoated 50 nm microvesicles was determined for five CNS regions of myelinated axons of adult rats comparing two methods of fixation. On the average, we found 1.3 microvesicular profiles per axon of which 9.4% were bristle coated. The proportion of microvesicles with coated profiles in myelinated axons was 1.2 to 2 times that observed among microvesicles of nerve terminals depending on fixation method. Relative abundance of coated vs. uncoated vesicles among various brain regions was not significantly different. The results demonstrate for the first time the presence of coated vesicles in axons other than at their terminals. Not only were vesicles present, but the relative proportions in terminal and somal portions were similar.     

Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols

Summary The effect of different fixation solutions on the denaturation of membrane-associated antigens in murine lymphoid cells was determined quantitatively using microfluorometric analysis and a radioimmunoassay. Paraformaldehyde and periodate-lysine-paraformaldehyde solutions preserved the antigenicity of cell surface-associated immunoglobulin (S–Ig) antigens when used in concentrations ranging from 0.01 to 4%. However, glutaraldehyde destroyed the antigenicity of S–Ig and Thy 1.2 molecules at concentrations higher than 0.1%. Electron microscopic analysis of the different fixed cell suspensions, after labelling of the cells with a rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (RaM-Ig-HRP) showed that prefixation of the sample with 0.1% glutaraldehyde was optimal for immunoelectron microscopical studies, since this concentration preserved both the antigenicity of membrane-associated antigens as well as the ultrastructure of the cells under study. Prolonged fixation periods affected antibody binding. However, S–Ig molecules denatured at a slower rate than Thy 1.2 molecules. A preparation method for the immunoelectron microscopical localization of lymphoid and non-lymphoid cell types in lymphoid organs is reported.TheHistochemical Journal lecture 1979 given by Dr Van Ewijk to the Histochemistry and Cytochemistry Section of the Royal Microscopical Society on 12 July, 1979.     

Comparison of LR white resin, Lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Summary Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Ultrastructure of Papillar Cells in Brassica campestris Revealed by Liquid Helium Rapid-Freezing and Substitution-Fixation Method

The ultrastructure of papillar cells of Brassica campestrison the day of anthesis was studied by the liquid helium rapid-freezingand a substitution-fixation method, abbreviated as the RFS method.Application of the RFS method to the analysis of papillar cellsenabled us to examine clear images of these cells which havenot been observed previously. The well-developed rough and smoothendoplasmic reticulum, numerous Golgi bodies and mitochondria,various small vesicles and clathrin-coated vesicles, were presentin the cells. The numbers of Golgi bodies, as well as the numbersof cisternae of each Golgi body, increased as compared to thatin the other cells of style. Lattice-like fenestrated and flattenedcisternae were seen adjacent to the narrowest trans cisternaof the Golgi body, which had a partially coated region at itsperiphery. Many coated vesicles were observed in the vicinityof this structure and the plasma membrane. Coated areas on theplasma membrane were also observed. The ultrastructure of papillarcells on the day of anthesis indicated that they are very activesecretory cells. By using an antibody against S₈-protein andsections prepared by the RFS method, we demonstrated the distributionof S₈-protein in the cell wall of papillar cells of homozygousplants of Brassica campestris S_gS₈. (Received June 26, 1990; Accepted September 29, 1990)     

Comparison of LR white resin,lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

Summary The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40°–50° C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

Identification of the coated vesicle proteins that bind calmodulin

The constituent proteins of coated vesicles responsible for binding calmodulin were identified by photoaffinity labeling with the reagent azido-¹²⁵I-calmodulin. Three protein complexes with apparent molecular weights of 130,000, 93,000 and 52,000 were labeled. Specificity was demonstrated by the dependence of labeling on Ca²⁺, and by its reduction in the presence of unlabeled calmodulin or Stelazine. Urea-soluble components of coated vesicles and material isolated by Sepharose CL4B chromatography formed a 52,000 MW labeled complex. Subtracting an apparent molecular weight of calmodulin of 20,000 from the weights of the covalently labeled complexes, the coated vesicle proteins that bind calmodulin are 110,000, 73,000 and 32,000 MW. The 32,000 MW protein is thought to participate in the coat structure but the other two are most likely associated with the vesicle.     

EGF induces receptor down-regulation with no receptor recycling in KB cells

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.     

Freeze-substitution improves the ultrastructural preservation of legume root hairs

The freeze-substitution method was applied toVicia hirsuta root hairs to test its effectiveness in improving preservation of the cell ultrastruture. Freeze-substitution almost certainly represents more faithfully the structure of the hair cell. A previously unreported ‘pyriformis’ vesicle is described. Also unique to freeze-substituted material are coated secretory vesicles; a smooth plasma membrane profile; mitochondrial ribosomes; long microfilament bundles which are associated with vesicles, mitochondria, coated pits and coated vesicles; microtubule-associated filaments; well-preserved coated vesicles and coated pits with enclosing rings; a pliciform nucleus. The results are discussed in context of previous reports using conventional fixation techniques.     

Formation of coated vesicles from coated pits in broken A431 cells

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti- transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.     

Immunogold localization of peroxisomal enzymes in Epon-embedded liver tissue. Enhancement of sensitivity by etching with ethanolic sodium hydroxide

Epon-embedded biological materials exhibit well preserved ultrastructure but generally weak antigenicity. Brief etching of ultrathin Epon sections with resin solvent, ethanolic sodium hydroxide, brought about a nearly 2-fold increase in the immunogold labeling density of rat and human hepatic peroxisomes after incubation with antisera against catalase and 3 enzymes of lipid beta-oxidation: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase. The etching was superior to pretreatment with an oxidant, sodium metaperiodate. Despite some deterioration of the cellular ultrastructure, the obtained enhancement of the sensitivity of the protein A-gold method may be helpful in cases when the antigenicity to be detected is strongly inhibited by epoxy resin embedding.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)     

An antibody against 100- to 116-kDa polypeptides in coated vesicles inhibits triskelion binding

There is considerable evidence that the 100- to 116-kDa polypeptides in calf brain coated vesicles are involved in the assembly of clathrin triskelions to form coated vesicles. We have raised polyclonal antibodies against these polypeptides. By Western blot analysis, these antibodies bind to a distinct subset of the six polypeptides in the region 100-116 kDa. Whole cell homogenates from calf brain, calf liver, and rat liver also show immunoreactivity in the 100-kDa region with no other cross reactivity. Isolated coated vesicles from calf liver, rat brain, and soybean roots also cross-react. Stripped coated vesicles, which are depleted of clathrin but which retain the 100- to 116-kDa polypeptides, quantitatively rebind 125I-triskelions. This binding is inhibited in a dose-dependent manner by 100- to 116-kDa antibody but not by nonimmune serum or by anti-clathrin polyclonal antibody. These studies indicate that (1) specific sites on the 100- to 116-kDa polypeptides are required for assembly of coated vesicles, and (2) this antibody will be useful in clarifying more precisely the role of the 100- to 116-kDa polypeptides in coated vesicle recycling.     

Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < or = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < or = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed >70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.     

Binding and internalization of gold-labeled IFN-gamma by human Raji cells

Binding and internalization of gold-labeled IFN-gamma (IFN-gamma/Au) by human Raji cells was examined by scanning and transmission electron microscopy. For SEM, visualization of gold particles was enhanced by the silver enhancement technique and by backscattered electron imaging. Binding studies revealed distinct labeling of microvilli-bearing cells after incubation with at least 10 U/ml IFN-gamma/Au, whereas cells with a smooth surface showed substantially lower labeling. After application of higher IFN-gamma (greater than 200 U/ml) concentrations, labeling intensity remained constant, which is consistent with the concentration of radiolabeled IFN-gamma required for saturating receptors on Raji cells. The specificity of IFN-gamma/Au binding was demonstrated by complete displacement with unlabeled IFN-gamma and by partial inhibition of labeling with a monoclonal anti-IFN-gamma R antibody. Thus, colloidal gold represents a valuable tag for visualizing the interaction of IFN-gamma with its receptor. Internalization of IFN-gamma/Au was initiated by accumulation of gold particles in coated pits which occurred within 10 min after warming of Raji cells. Additional incubation at 37 degrees C (up to 2 h) led to the appearance of gold particles in endocytic vesicles and lysosomes. Thus, our studies indicate that IFN-gamma/Au enters the Raji cells via the typical endocytotic pathway.