Immunocytochemical detection of fungi in the roots of Rhododendron

1986 ◽  
Vol 64 (4) ◽  
pp. 718-723 ◽  
Author(s):  
W. C. Mueller ◽  
B. J. Tessier ◽  
L. Englander
Keyword(s):  
Colloidal Gold ◽  
Thin Sections ◽  
Immunocytochemical Detection ◽  
Immunoglobin G

Antisera were prepared from fruiting structures of a Clavaria sp. found growing in the vicinity of Rhododendron plants and from mycelium of Pezizella ericae grown in culture. The antisera were used for the detection and differentiation of fungi in thin sections of roots of Rhododendron by an indirect immunocytochemical procedure using colloidal gold-labelled goat antirabbit immunoglobin G. Three distinct fungal types could be detected in roots: hyphae that reacted only with the Clavaria antiserum; hyphae that reacted only with the Pezizella antiserum; and hyphae that did not react with either antiserum.

scholarly journals Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes.

1986 ◽  
Vol 34 (7) ◽  
pp. 847-853 ◽  
Author(s):  
D R Abrahamson
Keyword(s):  
Colloidal Gold ◽  
Basement Membranes ◽  
Lamina Densa ◽  
Thin Sections ◽  
Gold Particles ◽  
Rabbit Igg ◽  
Gold Immunolocalization ◽  
Lowicryl K4m

Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.

scholarly journals Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin.

1989 ◽  
Vol 37 (2) ◽  
pp. 249-256 ◽  
Author(s):  
K Fujimoto ◽  
N Araki ◽  
K S Ogawa ◽  
S Kondo ◽  
T Kitaoka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Biologically Active ◽  
Myocardial Cells ◽  
Thin Sections ◽  
Gold Particles ◽  
Calmodulin Binding ◽  
Tissue Sections

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.

scholarly journals Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys.

1986 ◽  
Vol 103 (6) ◽  
pp. 2489-2498 ◽  
Author(s):  
D R Abrahamson ◽  
E W Perry
Keyword(s):  
Basement Membrane ◽  
Colloidal Gold ◽  
Early Stage ◽  
Basement Membranes ◽  
Newborn Rat ◽  
Thin Sections ◽  
Intravenous Injections ◽  
Rabbit Igg ◽  
Glomerular Development ◽  
Rat Kidneys

Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti-rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti-laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.

Colloidal Gold Markers: Preparation and Cell Labeling for Transmission and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 530-533
Author(s):  
Robert S. Molday
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Critical Evaluation ◽  
Cell Labeling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Backscattered Electrons ◽  
Wide Range ◽  
Scanning Electron

Colloidal gold particles have become one of the most widely used markers to detect, localize and, in some cases, quantitate cell surface and intracellular antigens and receptors since their introduction as transmission electron microscopic (TFM) markers by Faulk and Taylor in 1971 and as scanning electron microscopic (SEM) markers by Horisberger et al. in 1975. This interest in colloidal gold markers for cell labeling is based on their versatile properties for detection under the electron microscope. Colloidal gold particles are highly electron-dense which enables them to be seen under the TEM in thin sections of heavily stained cells. They can be prepared in a wide range of highly uniform sizes for visualization at different magnifications and for multiple labeling studies. Under the SEM, gold particles emit a high quantity of secondary electrons, backscattered electrons and characteristic X-ray signals and as a result, with the appropriate detectors, they can be readily distinguished from cell surface structures having a similar morphological appearance. The successful application of colloidal gold particles as markers for TEM and SEM however requires (i) careful preparation and characterization of both the gold markers and the ligand (protein)-gold conjugates, (ii) utilization of specific labeling techniques employing necessary controls to confirm the specificity of labeling, and (iii) critical evaluation of results in relation to the conditions used in labeling. These aspects of gold labeling will be considered here. Additional information can be obtained from recent reviews dealing specifically with gold markers and more generally with cell labeling techniques.

Characterization of beam-induced thinning and shrinkage of semi-thick sections in the HVEM

1990 ◽  
Vol 48 (3) ◽  
pp. 752-753
Author(s):  
J.R. Kremer ◽  
E.T. O'Toole ◽  
G.P. Wray ◽  
D.M. Mastronarde ◽  
S.J. Mitchell ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Colloidal Gold ◽  
Similar Data ◽  
Thin Sections ◽  
Gold Particles ◽  
Dose Rates ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope

It is well known that irradiation of plastic sections in a conventional transmission electron microscope (cTEM) causes specimen thinning and distortion. Thinning has been observed in the cTEM using several embedding media, using methods such as shrinkage of ordered paracrystalline structures, and shrinkage of sections coated with colloidal gold markers. The total thinning observed in the cTEM (80kev) is 30-50% for thin sections of epon araldite, but similar data do not exist for the HVEM at 1000 kev. Here we describe beam induced thinning and shrinkage of 0.2um sections in the HVEM.Experiments were performed using 0.2um sections of EPOX 812/Araldite or LX112 with 15 nm and 30 nm gold particles affixed to either surface of the section. The sections were initially tilted to approximately 25° and irradiated with known dose rates. Micrographs were taken at different times between 0-20 minutes then the sections were tilted back to 0° for a reference micrograph.

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 Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling.

1987 ◽  
Vol 35 (2) ◽  
pp. 229-231 ◽  
Author(s):  
H Morioka ◽  
M Tachibana ◽  
M Machino ◽  
A Suganuma
Keyword(s):  
Escherichia Coli ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Ultrastructural Localization ◽  
Polymyxin B ◽  
Thin Sections ◽  
Gold Particles ◽  
E Coli ◽  
Escherichia Coli Cells

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.

scholarly journals Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

1992 ◽  
Vol 40 (10) ◽  
pp. 1465-1470 ◽  
Author(s):  
D W Marshak
Keyword(s):  
Electron Microscopy ◽  
Tyrosine Hydroxylase ◽  
Colloidal Gold ◽  
Light And Electron Microscopy ◽  
Rabbit Antiserum ◽  
Gold Chloride ◽  
Thin Sections ◽  
Gold Particles ◽  
Rabbit Igg ◽  
Goldfish Retina

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.

scholarly journals Aminoglycoside binding sites in Escherichia coli as revealed by neomycin-gold labeling.

1986 ◽  
Vol 34 (7) ◽  
pp. 909-912 ◽  
Author(s):  
H Morioka ◽  
M Tachibana ◽  
T Amagai ◽  
A Suganuma
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Cytoplasmic Membrane ◽  
Thin Sections ◽  
Gold Particles ◽  
E Coli ◽  
Cytochemical Technique

A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli. Neomycin was conjugated chemically with bovine serum albumin (BSA). Colloidal gold was coated with the conjugated neomycin-BSA. The neomycin-BSA-gold was applied to thin sections of Epon-embedded E. coli and examined. Gold particles were observed on the outer membrane and the cytoplasmic membrane of E. coli. It was probably the ribosomes that were being labeled in the cytoplasm. Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.

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