Evaluation of blocking agents in protein A-gold immunocytochemistry

1988 ◽  
Vol 46 ◽  
pp. 386-387
Author(s):  
L. G. Komuves ◽  
E. J. King
Keyword(s):  
Plasma Membrane ◽  
Wheat Germ ◽  
Osmium Tetroxide ◽  
Protein A ◽  
L929 Cell ◽  
Blocking Agent ◽  
Gold Particles ◽  
Tissue Sections ◽  
Cacodylate Buffer ◽  
Low Levels

Various blocking agent solutions are used in protein A-gold immunocytochemistry to prevent or reduce nonspecific attachment of antibodies and/or protein A-gold particles to tissue sections. In our study of intracellular antigens present at low levels, the inadequacy of some commonly used blocking solutions became evident. We, therefore, compared several blocking solutions by exposing L929 cell cultures, at 4 °C, to a hapten-labeled lectin (dinitro-phenol-labeled wheat germ agglutinin, DNP-WGA) that binds specifically to the plasma membrane. We then used the protein A-gold method to locate the bound DNP-WGA. Since endocytosis had been prevented, gold particles not associated with the plasma membrane were regarded as nonspecific.The cells were incubated with DNP-WGA for 2 h at 4 °C, and were then fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. Some samples were also postfixed with osmium tetroxide.

scholarly journals Subcellular localization of cellulases in auxin-treated pea.

1976 ◽  
Vol 69 (1) ◽  
pp. 97-105 ◽  
Author(s):  
A K Bal ◽  
D P Verma ◽  
H Byrne ◽  
G A Maclachlan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Major Part ◽  
Osmium Tetroxide ◽  
Close Association ◽  
Cellulase Activity ◽  
Cytochemical Localization ◽  
Tissue Sections ◽  
Inner Surface

Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Gogli complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.

Immuno-gold post-embedding labeling for electron microscopy: Technical procedures and potential limitations

1990 ◽  
Vol 48 (3) ◽  
pp. 142-143
Author(s):  
Moise Bendayan
Keyword(s):  
Protein A ◽  
Cellular Structures ◽  
Protein G ◽  
Secondary Antibody ◽  
Antibody Molecule ◽  
Gold Particles ◽  
Tissue Sections ◽  
Antigenic Sites

The successful post-embedding immunolabeling of tissue sections requires several essential steps.1) The use of an highly specific and well characterized antibody.2) The use of an efficient probe that will bind the antibody molecule.3) The use of tissue sections prepared for microscopy which will combine good preservation of the cellular structures and retention of protein antigenicity.The characterization of the antibodies and their monospecificity are prerequisite conditions that will determine the final quality of the results.Several immunoprobes are available for the labeling of antigenic sites. The use of protein A or protein G, secondary antibody molecules tagged with colloidal gold particles or the biotin-avidin gold system, have been able to generate very good and reliable results of similar value. The question on the size of the gold particles remains a decision that should be taken according to the results expected. Labeling of large cellular structures and their visualization at low magnification require relatively large gold particles (10 to 15 nm).

scholarly journals Immunocytochemical localization of pectinesterases in hyphae of Phytophthora infestans

1987 ◽  
Vol 65 (12) ◽  
pp. 2607-2613 ◽  
Author(s):  
Helga Förster ◽  
Kurt Mendgen
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Phytophthora Infestans ◽  
Protein A ◽  
Immunocytochemical Localization ◽  
Hyphal Cell ◽  
Gold Particles ◽  
Different Types ◽  
Ultrathin Sections ◽  
Lowicryl K4m

Evidence for the localization of extracellular pectinesterases was obtained in hyphae of Phytophthora infestans with the antibody – protein A – gold technique. Hyphae were fixed in 1% formaldehyde and 0.5% glutaraldehyde, and the immunocytochemical localization was done on ultrathin sections of tissue embedded at low temperature in Lowicryl K4M. Gold particles were mainly present over three different types of vesicles and over dictyosomes. In older parts of the hyphae, the plasma membrane was heavily labeled. This might suggest that the hyphal cell wall in subapical regions is not as permeable as in the hyphal tips.

scholarly journals Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.

1985 ◽  
Vol 101 (2) ◽  
pp. 683-688 ◽  
Author(s):  
A Triller ◽  
F Cluzeaud ◽  
F Pfeiffer ◽  
H Betz ◽  
H Korn
Keyword(s):  
Plasma Membrane ◽  
Cervical Spinal Cord ◽  
Transmembrane Protein ◽  
Protein A ◽  
Microscopy Study ◽  
Ventral Horn ◽  
Immunofluorescent Staining ◽  
Antigenic Determinants ◽  
Glycine Receptors ◽  
Gold Particles

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.

THE DISTRIBUTION OF GP lb AND THE STABILITY OF THE PLASMA MEMBRANE ARE DEPENDENT UPON AN INTACT MEMBRANE SKELETON

1987 ◽  
Author(s):  
J K Boyles ◽  
JE B Fox ◽  
M C Berndt
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Protein A ◽  
Cell Aggregation ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Gold Particles ◽  
Actin Binding Protein ◽  
The Stability

Platelets are know to have a cytoskeleton of actin filaments. We have presented evidence that they also have a membrane skeleton linked to the cytoskeletal filaments and that the membrane skeleton is linked to GP Ib-IX on the plasma membrane via actin-binding protein. In the current study, electron microscopy of thick (0.2 ym) epoxy sections was used to identify the distribution of GP lb. After various treatments, platelets were fixed and incubated with affinity-purified GP lb antibody and colloidal gold-labeled Protein-A. The entire cell surface was covered with a network of short intersecting chains of relatively evenly spaced gold particles. This was true of platelets in blood dripped directly from a vein into fixative, of washed discoid platelets, and of platelets activated by thrombin, ionophore, or cold under conditions in which aggregation did not occur. This pattern was not affected by the size of the gold label, the immunocytochemical protocol, or the fixative. The number of gold particles per cell was between 10,000 and 20,000, indicating a 1:1 ratio of label to GP lb. The distribution of GP lb was not affected by a level of cyto-chalasin B sufficient to disrupt the actin filaments of the platelet cytoskeleton. Proteolysis of actin-binding protein is known to be induced by treatment of platelets with dibucaine and by platelet activation (with either ionophore or thrombin) under conditions in which cell aggregation occurs. These same treatments caused GP lb to cluster. They also produced platelets with unstable membranes that vesiculated when the cells were subjected to shear force during centrifugation or osmotic-ally stressed during fixation. These studies show that both the distribution of GP lb and membrane stability are dependent upon the integrity of actin-binding protein and the membrane skeleton. In the high-shear environment of the blood vessel, the membrane skeleton and its linkage to GP Ib-IX and the cytoskeleton may be essential for proper platelet function.

Ultrastructural Features of the Oral Apparatus of Stentor

1976 ◽  
Vol 34 ◽  
pp. 142-143
Author(s):  
Elizabeth F. Howell
Keyword(s):  
Plasma Membrane ◽  
Osmium Tetroxide ◽  
Buccal Cavity ◽  
Fiber Bundles ◽  
Microtubular Root ◽  
Stentor Coeruleus ◽  
Root Fiber

The ultrastructure of the normal oral apparatus of Stentor has not been extensively studied. I report here on the ultrastructure of the buccal cavity of Stentor coeruleus.Stentor coeruleus was fixed in either a buffered mixture of osmium tetroxide and glutaraldehyde, or in buffered glutaraldehyde alone. Cells were then dehydrated and embedded in a mixture of Epon and Araldite.An extensive adoral zone of membranelles surrounds the anterior of the cell, and each membranelle consists of 2 parallel rows of cilia. These extend down into the buccal cavity. Two microtubular root fibers, or nemadesmata (Figs. 2 and 5), extend deeply into the cytoplasm from the base of each ciliary kinetosome. Mitochondria are usually closely associated with the root fiber bundles, and small vesicles are present between the nemadesmata of adjacent kinetosomes (Fig. 5). In the cytopharyngeal, non-ciliated areas of the buccal cavity, microtubular ribbons which extend into the cytoplasm are aligned perpendicular to the plasma membrane of the buccal cavity (Figs. 1 and 2).

Oligodendrocytes in Developing Hameter Cerebral Cortex

1976 ◽  
Vol 34 ◽  
pp. 126-127
Author(s):  
MB. Tank Buschmann
Keyword(s):  
Cerebral Cortex ◽  
Optic Nerve ◽  
Frontal Cortex ◽  
Osmium Tetroxide ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Tissue Samples ◽  
Cacodylate Buffer ◽  
Layer V ◽  
Adult Hamster

Development of oligodendrocytes in rat corpus callosum was described as a sequential change in cytoplasmic density which progressed from light to medium to dark (1). In rat optic nerve, changes in cytoplasmic density were not observed, but significant changes in morphology occurred just prior to and during myelination (2). In our study, the ultrastructural development of oligodendrocytes was studied in newborn, 5-, 10-, 15-, 20-day and adult frontal cortex of the golden hamster (Mesocricetus auratus).Young and adult hamster brains were perfused with paraformaldehyde-glutaraldehyde in sodium cacodylate buffer at pH 7.3 according to the method of Peters (3). Tissue samples of layer V of the frontal cortex were post-fixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Epon-Araldite resin.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Opening images of synaptic vesicles and calcium localization by means of microwave fixation method

1990 ◽  
Vol 48 (2) ◽  
pp. 182-183
Author(s):  
Vinci Mizuhira ◽  
Hiroshi Hasegawa
Keyword(s):  
Synaptic Vesicles ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Sampling Procedure ◽  
Fixation Method ◽  
Potassium Oxalate ◽  
X Ray ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
Microwave Fixation

Microwave irradiation (MWI) was applied to 0.3 to 1 cm3 blocks of rat central nervous system at 2.45 GHz/500W for about 20 sec in a fixative, at room temperature. Fixative composed of 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4, also contained 2 mM of CaCl2 , 1 mM of MgCl2, and 0.1% of tannic acid for conventional observation; and fuether 30-90 mM of potassium oxalate containing fixative was applied for the detection of calcium ion localization in cells. Tissue blocks were left in the same fixative for 30 to 180 min after MWI at room temperature, then proceeded to the sampling procedure, after postfixed with osmium tetroxide, embedded in Epon. Ultrathin sections were double stained with an useal manner. Oxalate treated sections were devided in two, stained and unstained one. The later oxalate treated unstained sections were analyzed with electron probe X-ray microanalyzer, the EDAX-PU-9800, at 40 KV accelerating voltage for 100 to 200 sec with point or selected area analyzing methods.

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