Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

1989 ◽  
Vol 47 ◽  
pp. 1042-1043
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Calf Serum ◽  
Specific Protein ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Pass Through ◽  
Label Distribution ◽  
Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

scholarly journals GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.

1992 ◽  
Vol 40 (12) ◽  
pp. 1827-1835 ◽  
Author(s):  
S M Laurie ◽  
M B Mixon ◽  
J D Castle
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Reduced Form ◽  
Electron Microscopic Level ◽  
Apical Plasma Membrane ◽  
Cell Fractionation ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Common Component ◽  
Rat Parotid

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.

An electron microscopic study of vitellogenesis and egg membrane formation in Lytta nuttalli Say (ColeopterarMeloidae)

1970 ◽  
Vol 48 (4) ◽  
pp. 651-657 ◽  
Author(s):  
P. R. Sweeny ◽  
N. S. Church ◽  
J. G. Rempel ◽  
Wendy Frith
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Follicle Cells ◽  
Vitelline Membrane ◽  
Electron Microscopic ◽  
Yolk Formation ◽  
Membrane Formation ◽  
Electron Dense Material ◽  
Egg Membrane ◽  
Pass Through

Vitellogenesis and egg membrane formation in the terminal ovarian follicles of Lytta nuttatii were investigated by electron microscopy. Three kinds of yolk globules are produced. They apparently are composed predominantly of carbohydrates, lipids, and proteins, respectively. The "carbohydrate" and "lipid" yolk are assembled in the ooplasm, the former by rough endoplasmic reticulum and the latter by Golgi complexes. Their production begins early in oogenesis. "Proteid" yolk formation begins somewhat later. The "proteid" yolk globules evidently are formed from exfraovarian materials that pass through large spaces that develop between the follicular epithelial cells, then through the oocyte plasma membrane by pinocytosis. Fairly late in development, glycogen granules appear in the inner ooplasm. In the nearly fully grown follicle, the "membranous system" of the vitelline membrane is elaborated. It probably is formed largely from an electron-dense material of undetermined origin that accumulates outside the bases of the oocyte plasma membrane microvilli. Immediately after completion of the vitelline membrane, the chorion is laid down, presumably from dense globules of material produced by Golgi complexes in the follicle cells.

Quantitative Immunocytochemistry of Ca2+-Mg2+ ATPase in Ameloblasts Associated With Enamel Secretion and Maturation in the Rat Incisor

1996 ◽  
Vol 10 (2) ◽  
pp. 245-251 ◽  
Author(s):  
A.E. Zaki ◽  
A.R. Hand ◽  
M.I. Mednieks ◽  
D.R. Eisenmann ◽  
J.L. Borke
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Electron Microscopic ◽  
Rat Incisor ◽  
Enzyme Cytochemistry ◽  
Gold Particles ◽  
Early Maturation ◽  
Tissue Specimens ◽  
Immunohistochemical Techniques ◽  
Late Maturation

Our previous studies revealed intense membrane-associated labeling for Ca2+-Mg2+ ATPase (Ca2+-pump) in secretory and maturation ameloblasts in the rat incisor, both by enzyme cytochemistry and by immunohistochemical techniques. The purpose of the present study was to map the distribution of Ca2+-pump protein at the cellular and subcellular levels by means of a Ca2+-pump-specific monoclonal antibody and electron microscopic immunogold cytochemistry. Tissue specimens were dissected from secretory, early, and late enamel maturation zones. We quantified results by comparing gold particle densities over ameloblast lateral and distal plasma membrane regions, supranuclear cytoplasm, regions of the ruffled borders, and nuclei. The highest concentration of gold particles was seen over the distal membranes of early-maturation ameloblasts relative to those in late-maturation and secretory stages. Cytoplasmic labeling was less than that of the distal and lateral membranes, and gold particles located over nuclei were considered to be due to non-specific binding. These results are consistent with our earlier findings and suggest a role for the plasma membrane Ca2+-pump in the regulation of calcium availability to mineralizing enamel.

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 Localization of sugar-binding sites in Staphylococcus aureus using gold-labeled neoglycoprotein.

1994 ◽  
Vol 42 (12) ◽  
pp. 1609-1613 ◽  
Author(s):  
H Morioka ◽  
A Suganuma ◽  
M Tachibana
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Bacterial Strain ◽  
Wheat Germ ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Sugar Binding

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.

scholarly journals Preparation of colloidal gold-labeled agarose-gelatin microspherules for electron microscopic studies of phagocytosis in cultured cells.

1987 ◽  
Vol 35 (2) ◽  
pp. 163-173 ◽  
Author(s):  
K X Gao ◽  
L Huang
Keyword(s):  
Bovine Serum Albumin ◽  
Colloidal Gold ◽  
Room Temperature ◽  
Cultured Cells ◽  
Gold Chloride ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Egg Lecithin ◽  
Microscopic Studies ◽  
Span 80

Agarose-gelatin microspherules about 0.5 micron or larger are prepared with emulsification of 4% agarose-gelatin sol containing 0.2 M N-octylglucoside in an organic phase composed of cyclohexane, egg lecithin, Span 80, and ethanol, followed by extraction of lipophilic components with cyclohexane and ether. Colloidal gold particles are then introduced into microspherules using gold chloride reacting at room temperature with tannic acid in a specified concentration range. After they have been coated with bovine serum albumin or mouse IgG, colloidal gold-labeled microspherules can be readily phagocytized by mouse L-cells and P388 cells after incubation for several hours. In addition to their use as a novel marker for phagocytosis, we discuss other potential uses for these colloidal gold-labeled microspherules.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

A New Human Breast Tumor Cell Line

1975 ◽  
Vol 33 ◽  
pp. 388-389
Author(s):  
R.E. Nordquist ◽  
R.M. Wasik ◽  
P.J. Riggs ◽  
P.L. Munson ◽  
F.B. Schafer
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Calf Serum ◽  
Tumor Cell Line ◽  
Electron Microscopic Examination ◽  
Epithelial Cell Line ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Excised Tissue ◽  
Caucasian Female

An infiltrating ductal cell carcinoma was removed from the breast of a postmenopausal Caucasian female. The excised tissue was divided into three parts; one part for electron microscopy, one part for tissue culture and the remainder frozen for immunological studies.The tissue for culture was minced finely with sterile razor blades and cultured in Falcon flasks containing Eagel's MEM supplemented with 10% heat denatured fetal calf serum. The tissue for electron microscopy was fixed in 6.25% glutaraldehyde in 0.1 M PO4 buffer plus 5% sucrose and postfixed in 1% OsO4 in the same buffer. The fixed tissue was dehydrated in graded ethanol and embedded in Spurr.The tissue which was cultured began to grow out after approximately six weeks and became a continuous epithelial cell line which was designated BOT-2 (Breast Original Tumor). Electron microscopic examination revealed that these cells had epithelial characteristics, i.e. the presence of tonofilaments and well formed desmosomes.

Modified gach technique vs critical point drying: Shrinkage analysis for SEM

1987 ◽  
Vol 45 ◽  
pp. 954-955
Author(s):  
B. Thompson ◽  
N. Sculov ◽  
R.E. Crang
Keyword(s):  
Critical Point ◽  
Calf Serum ◽  
Drying Shrinkage ◽  
Specimen Preparation ◽  
Cell Shrinkage ◽  
Whole Cells ◽  
Critical Point Drying ◽  
Prepared Material ◽  
Inverted Microscope ◽  
Phosphate Buffered Saline

The use of co-polymerized glutaraldehyde-carbohydrazide (GACH) was proposed for specimen preparation in scanning electron microscopy (SEM) as a means of avoiding dehydration in organic solvents, and to provide dimensionally stable biological specimens through a process of air-drying. It has been assumed that shrinkage of specimens prepared by the GACH technique should be less than that of conventionally-prepared material by critical point drying (CPD). In a previous study, Bell has reported significant shrinkage of whole cells for SEM. This report compares cell shrinkage in GACH and CPD preparations.Fibroblasts from newborn rats were grown on collagen-coated glass cover-slips (with alpha numeric grids etched onto the surface of the coverslips) in Eagle's minimum essential medium + 10% fetal calf serum for 7 d. (3). Using an inverted microscope with phase-contrast optics, micrographs were taken of the cultures in their live state and 1 h. after fixation with 2.5% glutaraldehyde in Dulbecco's phosphate buffered saline (Figs. 1 and 3).

The use of 1nm silver enhanced gold probes for the detection of skin antigens

1990 ◽  
Vol 48 (3) ◽  
pp. 902-903
Author(s):  
J.P Cassella ◽  
H. Shimizu ◽  
A. Ishida-Yamamoto ◽  
R.A.J. Eady
Keyword(s):  
Type Iv Collagen ◽  
Freeze Substitution ◽  
Collagen Type Iv ◽  
Electron Microscopic ◽  
Normal Human Skin ◽  
Type Iv ◽  
Type Vii Collagen ◽  
Keratin Filaments ◽  
Normal Human ◽  
Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

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