Colloidal gold double labeling of two secretory proteins in bacterial cell

1983 ◽  
Vol 41 ◽  
pp. 614-615
Author(s):  
Guan Tinglu ◽  
Arati Ghosh ◽  
Bijan K. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Colloidal Gold ◽  
Colloidal Particles ◽  
Sodium Citrate ◽  
Secretory Proteins ◽  
Bacterial Cells ◽  
Double Labeling ◽  
Antibody Technique ◽  
Gold Particles ◽  
Multiple Labelling

Secretory proteins have been localized in bacterial cells by inmunoelectron-microscopy and cytochemical methods. Although ferritin-antibody technique is satisfactory for the localization of single antigenic sites, multiple labelling is not possible by this technique. However, such multiple labelling is possible using colloidal gold antibody because colloidal gold of widely varying sizes can be prepared by simple laboratory manipulation. We prepared colloidal gold of two different sizes and labelled these with anti-penicillinase and anti-alkaline phosphatase antibodies. These labels were used to determine the location of penicillinase and alkaline phosphatase in secreting Bacillus licheniformis 749/C cells.Large colloidal gold particles (150Å) were prepared by 10% sodium citrate treatment of 0.01% HAuCl4; the pH of this colloidal gold suspension was adjusted to 7.6 with 0.2 M K2CO3. Small colloidal particles (50A) were prepared by treating 0.01% HAuCl4, after pH was adjusted to 8.0 with 0.2M K2CO3, withphosphorous saturated ether. The large or small gold particles (1010 in 10 ml) were treated with 100μg of anti-alkaline phosphatase or anti-penicillinase antibodies; after thorough mixing unbound antibodies were removed by centrifugation.

scholarly journals Double immunocytochemical labeling applying the protein A-gold technique.

1982 ◽  
Vol 30 (1) ◽  
pp. 81-85 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Tissue Section ◽  
Protein A ◽  
Antigenic Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Gold Particles ◽  
Antigenic Sites

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

scholarly journals Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

1981 ◽  
Vol 89 (3) ◽  
pp. 653-665 ◽  
Author(s):  
H J Geuze ◽  
J W Slot ◽  
P A van der Ley ◽  
R C Scheffer
Keyword(s):  
Colloidal Gold ◽  
Protein A ◽  
Secretory Protein ◽  
The Other ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Fixed Tissue ◽  
Gold Particles ◽  
Polypeptide Backbone

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.

scholarly journals THE NUCLEAR ANNULI AS PATHWAYS FOR NUCLEOCYTOPLASMIC EXCHANGES

1962 ◽  
Vol 14 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Carl M. Feldherr
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Colloidal Gold ◽  
Colloidal Particles ◽  
Time Interval ◽  
Central Channel ◽  
Time Intervals ◽  
Gold Particles ◽  
Pass Through ◽  
Individual Particles

Colloidal gold particles, 25 to 55 A in diameter, which had been coated with polyvinylpyrrolidone, were microinjected into the ground cytoplasm of amebas (Chaos chaos). At time intervals of 1 minute, 2 minutes, 10 minutes, and 24 hours after injection the cells were fixed for electron microscopy. After 24 hours, gold particles were found in both the nuclei and the ground cytoplasm, the concentration being higher in the nuclei. Colloidal particles were also present in the nuclei after 10 minutes, but at this time interval the concentration did not appear to be greater than that in the ground cytoplasm. One and 2 minutes after injection, the gold particles were located almost exclusively in the ground cytoplasm; however, individual particles were often found within the annuli of the nuclear envelope, and were located specifically in the centers of these structures. The results suggest that at least some of the gold particles which enter the nuclei pass through the annuli, and that passage through these structures may be restricted to a central channel.

scholarly journals Sizing of protein A-colloidal gold probes for immunoelectron microscopy.

1981 ◽  
Vol 90 (2) ◽  
pp. 533-536 ◽  
Author(s):  
J W Slot ◽  
H J Geuze
Keyword(s):  
Colloidal Gold ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Pancreatic Tissue ◽  
Staphylococcal Protein A ◽  
Colloidal Solutions ◽  
Double Labeling ◽  
Gold Particles ◽  
Ultrathin Cryosections

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.

scholarly journals Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections.

1994 ◽  
Vol 42 (12) ◽  
pp. 1615-1623 ◽  
Author(s):  
T Takizawa ◽  
J M Robinson
Keyword(s):  
Human Neutrophil ◽  
Colloidal Gold ◽  
Model System ◽  
Protein Markers ◽  
Silver Enhancement ◽  
Double Labeling ◽  
Cytoplasmic Granules ◽  
Gold Particles ◽  
Ultrastructural Immunocytochemistry ◽  
Specific Granules

We present a new application for the use of small immunogold particles (approximately 1.4-nm diameter) for ultrastructural immunocytochemistry. These small gold particles have been used on ultra-thin cryosections in conjunction with a silver enhancement procedure that does not degrade ultrastructural detail. We have used the human neutrophil as a model system, in which known protein markers of two different cytoplasmic granules were localized, in the development of this procedure. The 1.4-nm immunogold particles coupled with silver enhancement yield intense labeling for localization of lactoferrin, a marker for the specific granules, and myeloperoxidase, a marker for the azurophil granules. Double labeling in which one antigen was visualized with 1.4-nm gold and silver enhancement and a second antigen was detected with colloidal gold-IgG on the same ultra-thin cryosection was successfully achieved. We also show that 1.4-nm diameter immunogold particles penetrate into cryosectioned neutrophils to a greater extent than 5-nm or 10-nm immunogold probes. These results show that small immunogold particles, along with silver enhancement, are a useful addition to the immunolabeling methods available for use with ultra-thin cryosections.

Synaptic circuitry of serotonin-synthesizing and serotonin-accumulating amacrine cells in the retina of the cane toad, Bufo marinus

1995 ◽  
Vol 12 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Bao-Song Zhu ◽  
Charles Straznicky ◽  
Ian Gibbins
Keyword(s):  
Colloidal Gold ◽  
Visual Information ◽  
Amacrine Cell ◽  
Phenylalanine Hydroxylase ◽  
Amacrine Cells ◽  
Bufo Marinus ◽  
Inner Plexiform Layer ◽  
Cane Toad ◽  
Double Labeling ◽  
Gold Particles

AbstractThe synaptic connections of amacrine cells synthesizing or accumulating serotonin in the retina of the cane toad, Bufo marinus, were studied by using preembedding double-labeling electron-microscopic immunocytochemistry. The binding sites of an anti-serotonin antibody were revealed by the diaminobenzidine reaction, whilst a colloidal gold-conjugated secondary antibody was used to detect an antibody to phenylalanine hydroxylase. Since the latter antibody recognizes tryptophan 5-hydroxylase, one of the synthesizing enzymes for serotonin, as well as tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, the double labeling of the present study enabled us to identify three groups of labeled profiles at the ultrastructural level. The profiles of serotonin-synthesizing amacrine cells contained both diaminobenzidine reaction product and colloidal gold particles, whilst those of serotonin-accumulating and dopaminergic amacrine cells contained only diaminobenzidine reaction product or colloidal gold particles, respectively. The synapses of serotonin-synthesizing or serotonin-accumulating amacrine cells were distributed all through the inner plexiform layer of the retina. The profiles of serotonin-synthesizing amacrine cells predominantly received synapses from, and made synapses onto, unlabeled amacrine cell dendrites. They also received synapses from, and made synapses onto, bipolar cell terminals. They also made synapses onto presumed ganglion cell dendrites. However, the profiles of serotonin-accumulating cells made synapses only with unlabeled amacrine cell processes. There were close contacts between the profiles of serotonin-synthesizing and serotonin-accumulating amacrine cells. No synaptic relationships were observed between dopaminergic and serotonin-synthesizing or serotonin-accumulating amacrine cells. These observations indicate that serotonin-synthesizing amacrine cells and serotonin-accumulating amacrine cells in the retina of Bufo marinus are involved in different microcircuits in visual information processing.

scholarly journals Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold.

1987 ◽  
Vol 35 (11) ◽  
pp. 1277-1284 ◽  
Author(s):  
R Jemmerson ◽  
M Agre
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Placental Alkaline Phosphatase ◽  
Gold Particles ◽  
Human Placental Alkaline Phosphatase ◽  
Membrane Components

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.

scholarly journals Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.

1984 ◽  
Vol 32 (7) ◽  
pp. 705-711 ◽  
Author(s):  
S Hisano ◽  
T Adachi ◽  
S Daikoku
Keyword(s):  
Colloidal Gold ◽  
Secretory Granules ◽  
Protein A ◽  
Freeze Substitution ◽  
Pituitary Cells ◽  
Tissue Preparation ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Cytoplasmic Matrix ◽  
Gold Particles

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.

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