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.

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 Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry.

1988 ◽  
Vol 36 (1) ◽  
pp. 107-109 ◽  
Author(s):  
S Yokota
Keyword(s):  
Particle Size ◽  
Quantitative Analysis ◽  
Rat Liver ◽  
Protein A ◽  
High Sensitivity ◽  
Thin Sections ◽  
Gold Particles ◽  
Effect Of Particle Size ◽  
Lowicryl K4m ◽  
Liver Peroxisomes

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.

scholarly journals Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.

1985 ◽  
Vol 100 (1) ◽  
pp. 118-125 ◽  
Author(s):  
J Roth ◽  
M J Lentze ◽  
E G Berger
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Thin Sections ◽  
Golgi Membranes ◽  
Basal Plasma ◽  
Basolateral Plasma Membrane ◽  
Junctional Complexes ◽  
Lowicryl K4m ◽  
Monospecific Antibodies ◽  
Absorptive Enterocytes

Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections

1979 ◽  
Vol 60 (3) ◽  
pp. 317-320 ◽  
Author(s):  
T. F. C. Batten ◽  
C. R. Hopkins
Keyword(s):  
Colloidal Gold ◽  
Protein A ◽  
Gold Particles ◽  
Ultrathin Sections

scholarly journals Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.

1989 ◽  
Vol 37 (9) ◽  
pp. 1353-1361 ◽  
Author(s):  
T Okami ◽  
A Yamamoto ◽  
K Omori ◽  
M Akayama ◽  
M Uyama ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aqueous Humor ◽  
Ciliary Body ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Immunoblot Analysis ◽  
Alpha Subunit ◽  
Ciliary Epithelium ◽  
Immunocytochemical Localization ◽  
Gold Particles

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.

scholarly journals Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 850-860 ◽  
Author(s):  
H Suzuki ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
K Tanoue ◽  
H Yamazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Induced Responses ◽  
Immunocytochemical Localization ◽  
Platelet Surface ◽  
Gold Particles ◽  
Fibrinogen Binding ◽  
Large Numbers ◽  
Lowicryl K4m

Abstract The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.

IMMUNOCYTOCHEMICAL LOCALIZATION OF FIBRINOGEN DURING THROMBIN-INDUCED AGGREGATION OF HUMAN PLATELETS

1987 ◽  
Author(s):  
H Suzuki ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
K Tanoue ◽  
H Yamazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Immunocytochemical Localization ◽  
Albumin Solution ◽  
Gold Particles ◽  
Gold Labelling ◽  
Lowicryl K4m ◽  
Induced Aggregation

The association of fibrinogen (Fbg) with washed platelets was studied during thrombin-induced aggregation in Tyrode-albumin solution with 2 mM Ca2+ or no added Ca2+. Platelets were fixed, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman Fbg, washed, reacted with gold-labelled rabbit anti-goat IgG and prepared for electron microscopy. To ensure that Fbg could be detected with this method, platelets were pretreated with chymotrypsin and aggregated with Fbg; gold particles were apparent on the surface and between adherent platelets and in the alpha granules. In a Ca2+-containing medium in the absence of external Fbg, washed platelets did not have Fbg on their surface although there was extensive gold labelling of the platelet alpha granules. Thrombin (0.05 U/ml) caused platelet aggregation, centralization and apparent fusion of alpha granules. By 60 sec large aggregates had formed, many platelets appeared degranulated but few gold particles were seen between adherent platelets. At 5 min, little Fbg remained in the aggregated platelets. In a few regions gold accumulated in fused granule material, and in occasional clusters between adherent platelets. In the presence of external Fbg (0.4 mg/ml) thrombin caused aggregation, centralization and fusion of granules, and discharge of granule contents. However, numerous gold particles were readily detectable between adherent platelets and on the platelet membrane. Fibrin formed and was abundantly labelled with immuno-gold. Similar findings were obtained in the medium without added Ca2+ (20 μM Ca2+). These results agree with observations obtained from measurements of 125I-Fbg binding and show that in the presence of external Fbg thrombin causes Fbg binding to platelets during aggregation. In the absence of added Fbg, thrombin aggregates platelets without extensive binding of released Fbg to the platelets.

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 In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.

1986 ◽  
Vol 102 (5) ◽  
pp. 1646-1653 ◽  
Author(s):  
M Binder ◽  
S Tourmente ◽  
J Roth ◽  
M Renaud ◽  
W J Gehring
Keyword(s):  
Electron Microscope ◽  
Protein A ◽  
Microscope Level ◽  
Gold Complex ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Hybrid Detection ◽  
Ultrathin Sections ◽  
Lowicryl K4m

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.

scholarly journals Pilocarpine-induced changes in the saccharide composition of the tectorial membrane and interdental cells of the organ of Corti: a study with gold-labeled lectins.

1994 ◽  
Vol 42 (3) ◽  
pp. 405-416 ◽  
Author(s):  
M E Rubio ◽  
J Rueda ◽  
J J Prieto ◽  
J A Merchán
Keyword(s):  
Low Temperature ◽  
Protein A ◽  
Organ Of Corti ◽  
Tectorial Membrane ◽  
Systemic Injection ◽  
Gold Particles ◽  
Lowicryl K4m ◽  
Induced Changes ◽  
Labeling Pattern ◽  
Number Of Particles

The glycoconjugates in the cytoplasm of inner ear interdental cells and those constituting the limbal tectorial membrane were identified by a post-embedding cytochemical method using low-temperature embedding in Lowicryl K4M and labeling with biotinylated lectins, goat anti-biotin antibody, rabbit anti-goat antibody, and gold-labeled protein A in control animals, and after the systemic injection of pilocarpine. The lectins used were ConA, PHA-E, PSA, RCA, SBA, Succ-WGA, UEA, and WGA. In control animals, a semiquantitive analysis of gold particles showed that Succ-WGA produced the strongest labeling on the tectorial membrane, followed by SBA, ConA, WGA, RCA, PHA-E, and PSA. The lowest values were obtained with UEA. The cytoplasm of the interdental cells was also labeled with all the lectins, but the number of particles/microns2 was lower than on the tectorial membrane. The concentration of gold particles on the limbal tectorial membrane in pilocarpine-treated animals was higher than in control animals for some lectins (RCA, PSA, UEA) but lower for others (WGA, SBA, PHA-E, Succ-WGA). The changes in the labeling pattern of the cytoplasm of the interdental cells paralleled those in the tectorial membrane. These results demonstrate that the saccharide composition of the limbal tectorial membrane can be modified by systemic injection of pilocarpine. This action may take place through a change in either the secretion rate or the amount of some glycoconjugates by the interdental cells.

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