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 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 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 Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

1995 ◽  
Vol 43 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D Malide ◽  
N G Seidah ◽  
M Chrétien ◽  
M Bendayan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Endoproteolytic Cleavage

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

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 Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya.

1985 ◽  
Vol 33 (6) ◽  
pp. 499-507 ◽  
Author(s):  
R Lamberts ◽  
P C Goldsmith
Keyword(s):  
Immunoglobulin G ◽  
Colloidal Gold ◽  
Gold Deposition ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Hypothalamic Neurons ◽  
Double Labeling ◽  
Thin Sections ◽  
Beta Endorphin ◽  
Rabbit Immunoglobulin

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.

scholarly journals Freeze-drying technique in electron microscopic immunohistochemistry.

1985 ◽  
Vol 33 (5) ◽  
pp. 485-490 ◽  
Author(s):  
S Hisano ◽  
T Adachi ◽  
S Daikoku
Keyword(s):  
Freeze Drying ◽  
Secretory Granules ◽  
Protein A ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Axon Terminals ◽  
Conventional Procedure ◽  
Small Gold Particle ◽  
Excellent Preservation ◽  
Gold Label

Postembedding immunocytochemical labeling was performed on sections of rat neurohypophysis prepared by either freeze-drying, vapor fixation and Spurr resin embedding, or conventional aqueous fixation and Spurr resin embedding. Arginine vasopressin (AVP) and oxytocin (OXT) were immunolabeled with protein A-gold-anti-AVP and protein A-gold-anti-OXT complexes, respectively. The freeze-drying procedure (FD) resulted in excellent preservation of ultrastructure and greater antigenicity than the conventional procedure (Con). More gold particles were seen over secretory granules in FD sections than in Con sections. In addition, in FD sections, the gold label was restricted to secretory granules while in Con sections, both the granules and the extragranular axoplasm exhibited label. The two antigens in FD sections could be labeled simultaneously with protein A-small gold particle-anti-OXT complex and protein A-large gold particles-anti-AVP complex. In this way the two antigens were seen to be present in secretory granules within different axon terminals. Thus FD preparations should be useful for demonstrating the presence of multiple antigens in the same granules of nerve terminals.

Ultrastructural Aspects of Golgi Complex Function in Parathyroid Cells of Winter Frogs

1973 ◽  
Vol 31 ◽  
pp. 680-681
Author(s):  
William J. Dougherty
Keyword(s):  
Golgi Complex ◽  
Secretory Granules ◽  
Complex Function ◽  
Secretory Activity ◽  
Secretory Proteins ◽  
Perivascular Spaces ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Ca Ions ◽  
Regulation Of Secretion

The regulation of secretion in exocrine and endocrine cells has long been of interest. Electron microscopic and other studies have demonstrated that secretory proteins synthesized on ribosomes are transported by the rough ER to the Golgi complex where they are concentrated into secretory granules. During active secretion, secretory granules fuse with the cell membrane, liberating and discharging their contents into the perivascular spaces. When secretory activity is suppressed in anterior pituitary cells, undischarged secretory granules may be degraded by lysosomes. In the parathyroid gland, evidence indicates that the level of blood Ca ions regulates both the production and release of parathormone. Thus, when serum Ca is low, synthesis and release of parathormone are both stimulated; when serum Ca is elevated, these processes are inhibited.

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 Localization of ligands for L-selectin in mouse peripheral lymph node high endothelial cells by colloidal gold conjugates

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3766-3775 ◽  
Author(s):  
A Kikuta ◽  
SD Rosen
Keyword(s):  
Endothelial Cells ◽  
Lymph Node ◽  
Colloidal Gold ◽  
Secretory Granules ◽  
Polyclonal Antiserum ◽  
Secretory Product ◽  
Electron Microscopic ◽  
High Endothelial Venules ◽  
Ultrastructural Studies ◽  
Peripheral Lymph

L-selectin, a Ca(2+)-dependent lectin-like receptor, mediates lymphocyte attachment to high endothelial venules (HEV) of peripheral lymph nodes (PLN) during the process of lymphocyte homing. Two endothelial-derived ligands for L-selectin, known as GlyCAM-1 (Sgp50) and CD34 (Sgp90), have been identified by affinity precipitation of lymph node extracts with a chimeric molecule that combines the extracellular domains of L-selectin with the human IgG1 Fc region (L- selectin-IgG) (J Cell Biol 110:2221, 1990). Here, using a histologic probe based on colloidal gold conjugated to L-selectin-IgG (LS-Ig), we performed morphologic mapping of the HEV ligands in PLN at both the light and electron microscopic levels. With a postembedding labeling method, intense LS-Ig-gold staining of PLN HEV was observed, while the HEV of Peyer's patches (PP) were negative. The specificity of LS-Ig- gold staining was established by pretreatment of sections with sialidase and coincubation of sections with EGTA, fucoidin, or L- selectin-IgG itself. In ultrastructural studies of high endothelial cells(HEC), gold particles were bound to the trans-Golgi network(TGN) and to peripheral vesicles in the cytoplasm. Gold labeling was also detected in a patchy distribution on the entire luminal vascular surface of HEC. Although the perivascular fibroreticular sheath of HEV was frequently labeled limited labeling was observed on the basolateral surfaces of the HEC. In most cases, the HEC membrane surrounding migrating lymphocytes was negative. These results show that L-selectin ligands pass through the Golgi apparatus during their biosynthesis, are stored in secretory granules, and are expressed on the vascular luminal surface of the HEC. A polyclonal antiserum to GlyCAM-1 intensely stained intracellular organelles in the biosynthetic pathway including cytoplasmic vesicles, but failed to stain the cell surface of HEC. Given its presence in serum as a soluble factor, GlyCAM-1 is likely to be a secretory product.

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