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.

scholarly journals Preparation of Protein a Gold

1997 ◽  
Vol 5 (5) ◽  
pp. 12-13
Author(s):  
Paul Webster
Keyword(s):  
Electron Microscopy ◽  
Biological Activity ◽  
Colloidal Gold ◽  
Protein A ◽  
List Type ◽  
Type Number ◽  
Stained Glass ◽  
Small Particles ◽  
Silver Enhancement ◽  
Gold Particles

Colloidal gold has been used for centuries in the preparation of stained glass for windows and fine glassware. In recent years, colloidal gold particles have become a useful tool in microscopy for staining tissues and sections. Colloidal gold particles are especially useful for biological electron microscopy, Some of the reasons why are listed below.*Homogeneous preparations of particles varying in size from 3μm to 20μm can be easily prepared.*Colloidal gold suspensions are inexpensive to prepare. Most proteins can be easily coupled to colloidal gold particles.*Most proteins can be easily coupled to colloidal gold particles.*Proteins coupled to gold particles do not appear to lose their biological activity.*The colloidal gold particles can be easily seen in the electron microscope.*Colloidal gold does not naturally occur in biological material. Therefore, if you see it, it is because you put it there.*Colloidal gold probes can be used for light microscopy, The larger gold particles can be directly observed by the light microscope. Small particles are detected by silver enhancement or epipolarized illumination.*The same probes can be used for both LM and TEM imrnunocytochemistry.

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.

Novel labeling methods for em analysis of ultrathin cryosections

1996 ◽  
Vol 54 ◽  
pp. 894-895
Author(s):  
John M. Robinson ◽  
Toshihiro Takizawa
Keyword(s):  
Colloidal Gold ◽  
Model System ◽  
Human Neutrophils ◽  
Frozen Sections ◽  
Specific Granules ◽  
Gold Label ◽  
Ultrathin Cryosections ◽  
Ultrathin Frozen Sections ◽  
Particulate Probes ◽  
Enzyme Alkaline Phosphatase

Ultrathin cryosections are the most favorable material for localization of intracellular antigens with particulate probes (e.g., colloidal gold) in post-embedding immunocytochemistry. Cryosections are prepared under the most benign conditions as compared to embedding samples in various kinds of plastic media. Typically, higher labeling efficiencies can be achieved with ultrathin frozen sections than with plastic sections.We have utilized human neutrophils, the most abundant type of leukocyte, as a model system to explore labeling procedures for ultrathin cryosections. These labeling procedures can serve as alternative or complementary approaches to the traditional colloidal gold label. Neutrophils are characterized by the presence of numerous granules in their cytoplasm. For many years, the model for neutrophil structure has held that there are two granule types in these cells, the socalled azurophilic and specific granules. We have described an additional cytoplasmic compartment with unusual properties in neutrophils. This compartment is characterized by the presence of the enzyme alkaline phosphatase (Fig. 1).

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 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 Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy

1997 ◽  
Vol 45 (5) ◽  
pp. 631-642 ◽  
Author(s):  
John M. Robinson ◽  
Dale D. Vandré
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
Model System ◽  
The Other ◽  
Secondary Antibody ◽  
Correlative Microscopy ◽  
Silver Enhancement ◽  
Fab Fragment ◽  
Gold Particles

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

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