Immunocytochemical localization of enterobacterial common antigen in Escherichia coli and Yersinia enterocolitica cells.

Enterobacterial common antigen (ECA) was localized on Lowicryl K4M sections and on ultrathin cryosections by using either a mouse monoclonal antibody or an absorbed rabbit polyclonal immune serum with the corresponding gold-labeled secondary antibodies. Comparable results were obtained with both monoclonal antibody and polyclonal immune serum. Controls with two ECA-negative mutants revealed the ECA specificity of both labeling systems. On Lowicryl K4M sections, good labeling of the outer membrane and of membrane-associated areas in the cytoplasm was obtained. Unexpectedly, however, the ribosome-containing areas of the cytoplasm also showed significant labeling. On ultrathin cryosections, labeling of the cytoplasmic areas was much weaker, although the density of label in the outer membrane was comparable to that obtained with the Lowicryl K4M sections. With the techniques used, it cannot be completely excluded that the appearance of ECA in the cytoplasm is due to displacement of ECA-reactive sites during the preparation procedure.     

Specificity of an Antiserum Produced Against Pseudomicrothorax dubius Trichocysts Prepared by a Rapid Isolation Method

ABSTRACT. A rapid method was developed for the isolation of Pseudomicrothorax dubius ciliary and trichocyst fractions which were characterized by SDS-PAGE followed by combined silver and Coomassie blue staining. Antibodies were prepared against the trichocyst fraction and employed to label Lowicryl thin sections of cells. Trichocysts were strongly labeled, as were the surfaces of the plasma and ciliary membranes. Immunoblots of the trichocyst fraction revealed labeling of major bands at 16–29 kD, characteristic of the trichocyst proteins. On immunoblots of the ciliary fraction, approximately eight bands were labeled, including the major cell surface glycoprotein, the immobilization antigen. Ciliary proteins not located on the membrane surface, such as the tubulins, were not labeled. Absorption of the antiserum against fixed P. dubius cells eliminated the cell surface labeling on Lowicryl sections and on immunoblots of the ciliary fraction. The major trichocyst protein bands were as strongly labeled as with the nonabsorbed antiserum. Labeling of several of the minor, higher molecular weight bands of the trichocyst fraction was eliminated, indicating that they are cell surface contaminants. Of the two major structural components of the trichocyst, the shaft and the arms, the antiserum is shown to react nearly exclusively with the shaft proteins on both Lowicryl sections and immunoblots.     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

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

Prevention of non-specific interactions of gold-labeled reagents on tissue sections

The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling. We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling. We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells

In an effort to understand the regulation of the transition of a mature neuron to the growth, or regenerating, state we have analyzed the composition of the axonally transported proteins in the retinal ganglion cells of the toad Bufo marinus after inducing axon regeneration by crushing the optic nerve. At increasing intervals after axotomy, we labeled the retinal ganglion cells with [35S]methionine and subsequently analyzed the labeled transported polypeptides in the crushed optic nerve by means of one- and two-dimensional electrophoretic techniques. The most significant conclusion from these experiments is that, while the transition from the mature to the regenerating state does not require a gross qualitative alteration in the composition of axonally transported proteins, the relative labeling of a small subset of rapidly transported proteins is altered dramatically (changes of more than 20-fold) and reproducibly (more than 30 animals) by axotomy. One of these growth-associated proteins (GAPs) was soluble in an aqueous buffer, while three were associated with a crude membrane fraction. The labeling of all three of the membrane-associated GAPs increased during the first 8 d after axotomy, and they continued to be labeled for at least 4 wk. The modulation of these proteins after axotomy is consistent with the possibility that they are involve in growth-specific functions and that the altered expression of a small number of genes is a crucial regulatory event in the transition of a mature neuron to a growth state. In addition to these selective changes in rapidly transported proteins, we observed the following more general metabolic correlates of the regeneration process: The total radioactive label associated with the most rapidly transported proteins (groups I and II) increased three to fourfold during the first 8 d after the nerve was crushed, while the total label associated with more slowly moving proteins (group IV) increased about 10-fold during this same period. Among these more slowly transported polypeptides, five were observed whose labeling increased much more than the average. Three of these five polypeptides resemble actin and alpha- and beta-tubulin in their electrophoretic properties.     

Localization of lectin-binding sites and sugar-binding proteins in tachyzoites of Toxoplasma gondii

Lectins and neoglycoproteins labeled with colloidal gold particles were used for the ultrastructural localization of carbohydrate residues and sugar-binding sites, respectively, in thin sections of tachyzoites of Toxoplasma gondii embedded in the Lowicryl K4M resin. Incubation of the sections in the presence of gold-labeled Canavalia ensiformis (Con A), Arachis hypogaea (PNA), Ricinus communis I (RCA I), Triticum vulgaris (WGA), and Limax flavus (LFA) agglutinins showed significant labeling of the rhoptries. However, no labeling of the parasite's surface was observed. Incubation of tachyzoites in the presence of gold-labeled albumin-N-acetyl-D-glucosamine or albumin-galactose, but not in the presence of albumin-mannose, led to labeling of the rhoptries in a pattern similar to that observed with the lectins. The results obtained are discussed in relation to the possible role played by secretion of rhoptry macromolecules during the process of T. gondii-host cell interaction.     

Immunogold localization of nitrate reductase in maize leaves

Mature maize leaf tissue (Zea mays L.) was immunolabeled using a pre-embedding protocol with specific antibodies for nitrate reductase and protein A-colloidal gold. Immunogold label was found exclusively in the cytoplasm of mesophyll cells; no reaction was detected in bundle sheath cells. Chloroplasts, which were sliced open during cryosectioning, had no labeling. Thus, it appears nitrate reductase is localized exclusively in the cytoplasm of maize leaf mesophyll cells. No gold labeling was found on tissue sections embedded in L. R. White's or Lowicryl resin, indicating that previous chloroplast localization utilizing these protocols may be artifactual, resulting from shared epitopes or nonspecific antibody binding.     

Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.     

Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens

Baby hamster kidney cells infected with Semliki Forest virus were used as a model system for quantitative immunocytochemical labeling studies. In this system, a well-characterized membrane protein complex is present in different concentrations in three separate locations. Using immunogold labeling of cryosections, we compared the number of gold particles labeling the membranes of endoplasmic reticulum, Golgi stack, and fully formed virions at the plasma membrane to the biochemically determined concentrations. The efficiency of labeling was 40, 13, and 14% for the three structures, respectively. In a comparative study, Lowicryl K4M sections were found to give significantly lower levels of labeling.     

Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.     

An integral membrane glycoprotein associated with an endocytic compartment of Trypanosoma vivax: identification and partial characterization

A 65-kD glycoprotein (gp65) of Trypanosoma (Duttonella) vivax was identified using a murine monoclonal antibody (mAb 4E1) that had been raised against formalin-fixed, in vitro-propagated, uncoated forms. Intracellular localization studies utilizing the mAb in immunofluorescence on fixed, permeabilized T. vivax bloodstream forms and immunoelectron microscopy on thin sections of Lowicryl K4M-embedded cells revealed labeling of vesicles and tubules in the posterior portion of the parasite. Some mAb-labeled vesicles contained endocytosed 10 nm BSA-gold after incubation of the parasites with the marker for 5-30 min at 37 degrees C, and the greatest degree of colocalization was observed after 5 min. Double labeling experiments using the mAb and a polyclonal anti-variant surface glycoprotein (VSG) antibody to simultaneously localize both gp65 and VSG demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways within the cell. Using the mAb for immunoblot analyses, gp65 was shown to be enriched in a fraction of solubilized membrane proteins eluted from either immobilized Con A or Ricinus communis agglutinin and was found to possess carbohydrate linkages cleaved by both endoglycosidase H and O-glycosidase, suggesting the presence of N- and O-linked glycans. Protease protection and crosslinking experiments suggest that gp65 is a transmembrane protein with trypsin cleavage and NH2-crosslinking sites on the lumenal face of the vesicles.     

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.     

Secretory proteins in a ciliate cell: Identification of epitopes common to numerous polypeptides

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: I) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15–19, 22 and 24 kDa, pI 4.6?6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0?5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.