scholarly journals Intragranular colocalization of immunoreactive methionine-enkephalin and oxytocin within the nerve terminals in the posterior pituitary.

1985 ◽  
Vol 33 (9) ◽  
pp. 891-899 ◽  
Author(s):  
T Adachi ◽  
S Hisano ◽  
S Daikoku
Keyword(s):  
Colloidal Gold ◽  
Secretory Granules ◽  
Protein A ◽  
Complex Method ◽  
Nerve Terminals ◽  
Tissue Preparation ◽  
Methionine Enkephalin ◽  
Gold Particles ◽  
Tissue Antigens ◽  
Antibody Complex

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.

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.

scholarly journals Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1069-1074 ◽  
Author(s):  
S Akagi ◽  
A Yamamoto ◽  
T Yoshimori ◽  
R Masaki ◽  
R Ogawa ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Amorphous Material ◽  
Protein A ◽  
Disulfide Isomerase ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Acinar Lumen ◽  
Protein Disulfide

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

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.

scholarly journals Selective binding, uptake, and retrograde transport of tetanus toxin by nerve terminals in the rat iris. An electron microscope study using colloidal gold as a tracer

1978 ◽  
Vol 77 (1) ◽  
pp. 1-13 ◽  
Author(s):  
ME Schwab ◽  
H Thoenen
Keyword(s):  
Electron Microscope ◽  
Tetanus Toxin ◽  
Colloidal Gold ◽  
Smooth Endoplasmic Reticulum ◽  
Specific Binding ◽  
Retrograde Transport ◽  
Nerve Terminals ◽  
Gold Complexes ◽  
Selective Binding ◽  
Gold Particles

A series of specific macromolecules (tetanus toxin, cholera toxin, nerve growth factor [NGF], and several lectins) have been shown to be transported retrogradely with high selectivity from terminals to cell bodies in various types of neurons. Under identical experimental conditions (low protein concentrations injected), most other macromolecules, e.g. horseradish peroxidase (HRP), albumin, ferritin, are not transported in detectable amounts. In the present EM study, we demonstrate selective binding of tetanus toxin to the surface membrane of nerve terminals, followed by uptake and subsequent retorgrade axonal transport. Tetanus toxin or albumin was adsorbed to colloidal gold particles (diam 200 A). The complex was shown to be stable and well suited as an EM tracer. 1-4 h after injection into the anterior eye chamber of adult rats, tetanus toxin-gold particles were found to be selectively associated with membranes of nerve terminals and preterminal axons. Inside terminals and axons, the tracer was localized mainly in smooth endoplasmic reticulum (SER)-like membrane compartments. In contrast, association of albumin-gold complexes with nervous structures was never observed, in spite of extensive uptake into fibroblasts. Electron microscope and biochemical experiments showed selective retrograde transport of tetanus toxin-gold complexes to the superior cervical ganglion. Specific binding to membrane components at nerve terminals and subsequent internalization and retrograde transport may represent an important pathway for macromolecules carrying information from target organs to the perikarya of their innervating neurons.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

scholarly journals Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

1991 ◽  
Vol 39 (6) ◽  
pp. 809-821 ◽  
Author(s):  
S Tanaka ◽  
M Nomizu ◽  
K Kurosumi
Keyword(s):  
Polyclonal Antibody ◽  
Secretory Granules ◽  
Protein A ◽  
Proteolytic Processing ◽  
Pars Intermedia ◽  
Gold Particles ◽  
Melanocyte Stimulating Hormone ◽  
Rat Pituitary ◽  
Almost All ◽  
Electron Lucent

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.

Characterization of an optimal protein A-gold complex: Improvement of the labeling efficiency

1990 ◽  
Vol 48 (3) ◽  
pp. 942-943
Author(s):  
Lucian Ghitescu ◽  
Moise Bendayan
Keyword(s):  
Polyethylene Glycol ◽  
Gold Particle ◽  
Colloidal Gold ◽  
Specific Activity ◽  
Protein A ◽  
Gold Complex ◽  
Dense Layer ◽  
Gold Particles ◽  
Optical Absorbance

By using 125I protein A, we have prepared complexes of this immunoprobe with colloidal gold particles having diameters ranging from 5 to 15 nm. Various concentrations of protein A (from 35 to 1000 nM) of known specific activity were employed in order to obtain a spectrum of conjugates differing not only in sizes, but also in amounts of protein A they carry. After ultracentrifugation through a step gradient (15, 30 and 70% w/v) of sucrose, the protein A-gold complexes were quantitatively recovered from the most dense layer, and diluted in a 0.01 M phosphate buffer containing 0.01% polyethylene glycol. The conjugates did not contain free protein A, as proven by the lack of radioactivity in the intermediate sucrose layers. By correlating the radioactivity of the complexes with their particle densities (inferred from the optical absorbance at 520 nm), the number of protein A molecules bound per gold particle was calculated for each experimental condition.

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.

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