Immuno-electron microscopic identification of Methanosarcina spp. in anaerobic digester fluid

1985 ◽  
Vol 31 (9) ◽  
pp. 839-844 ◽  
Author(s):  
Ralph W. Robinson ◽  
Gregory W. Erdos
Keyword(s):  
Protein A ◽  
Methanobacterium Thermoautotrophicum ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Methanosarcina Mazei ◽  
Pure Cultures ◽  
Electron Microscopic Identification ◽  
Mixed Samples ◽  
Heavy Labelling

Whole cell antibodies against Methanosarcina mazei strain S6 were used with protein A – colloidal gold to identify bacteria in thin sections of samples from anaerobic methane producing digesters. It was possible to identify bacteria at the genus level and to show relatedness at the species and strain levels. Heavy labelling was observed on thin sections of the immunogenic strain S6. Lighter labelling was observed with pure cultures of M. mazei strain LYC. Pure cultures of Methanococcus voltae or Methanobacterium thermoautotrophicum did not label. In samples from mesophilic sewage digesters, sarcinal colonies of bacteria similar to Methanosarcina showed heavy labelling per cell while colonies from 55 °C digesters show lighter labelling. A few free cocci, which were released from the sarcinal colonies, also labelled. Labelling was not observed on other bacterial forms in either set of digesters. These studies indicate that a collection of bacterial antibodies can be used to identify and map bacteria in situ in mixed samples.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

1992 ◽  
Vol 40 (11) ◽  
pp. 1647-1657 ◽  
Author(s):  
R W Dirks ◽  
A G Van Dorp ◽  
J Van Minnen ◽  
J A Fransen ◽  
M Van der Ploeg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Lymnaea Stagnalis ◽  
Pond Snail ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Double Labeling ◽  
Thin Sections ◽  
Cell Hormone

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

scholarly journals Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.

1986 ◽  
Vol 34 (2) ◽  
pp. 189-195 ◽  
Author(s):  
S A Ernst ◽  
J R Palacios ◽  
G J Siegel
Keyword(s):  
Choroid Plexus ◽  
Epithelial Transport ◽  
Protein A ◽  
Fluid Secretion ◽  
Physiological Data ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Membrane Surfaces

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.

scholarly journals The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles.

1980 ◽  
Vol 84 (3) ◽  
pp. 513-530 ◽  
Author(s):  
S Tsukita ◽  
H Ishikawa
Keyword(s):  
Structural Integrity ◽  
Smooth Endoplasmic Reticulum ◽  
Retrograde Transport ◽  
Radioactive Tracers ◽  
Local Cooling ◽  
Electron Microscopic ◽  
Unmyelinated Axons ◽  
Uniform Diameter ◽  
Electron Microscopic Identification

To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction.

scholarly journals Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique.

1987 ◽  
Vol 35 (7) ◽  
pp. 795-801 ◽  
Author(s):  
S A Hearn
Keyword(s):  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Neuroendocrine Cells ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Carotid Body Tumors

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.

scholarly journals In situ ATR-IR spectroscopic and electron microscopic analyses of settlement secretions of Undaria pinnatifida kelp spores

2010 ◽  
Vol 8 (56) ◽  
pp. 410-422 ◽  
Author(s):  
L. Petrone ◽  
R. Easingwood ◽  
M. F. Barker ◽  
A. J. McQuillan
Keyword(s):  
Ir Spectroscopy ◽  
Sea Water ◽  
Undaria Pinnatifida ◽  
Bulk Material ◽  
Attenuated Total Reflection ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Electron Microscopies ◽  
Anionic Polysaccharides

Knowledge about the settlement of marine organisms on substrates is important for the development of environmentally benign new methods for control of marine biofouling. The adhesion to substrates by spores of Undaria pinnatifida , a kelp species that is invasive to several countries, was studied by scanning electron and transmission electron microscopies (SEM/TEM) as well as by in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The IR spectra showed that adhesive secretion began approximately 15 min after initial settlement and that the adhesive bulk material contained protein and anionic polysaccharides. Energy dispersive X-ray microanalysis of the adhesive identified sulphur and phosphorus as well as calcium and magnesium ions, which facilitate the gelation of the anionic polysaccharides in the sea water. The adhesive may be secreted from Golgi bodies in the spore, which were imaged by TEM of spore thin sections. Additionally, an in situ settlement study on TiO 2 particle film by ATR-IR spectroscopy revealed the presence of phosphorylated moieties directly binding the substrate. The presence of anionic groups dominating the adhesive suggests that inhibition of spore adhesion will be favoured by negatively charged surfaces.

scholarly journals Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.

1982 ◽  
Vol 30 (7) ◽  
pp. 691-696 ◽  
Author(s):  
J Roth
Keyword(s):  
Protein A ◽  
Second Step ◽  
Staphylococcal Protein A ◽  
Paraffin Sections ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Antigen Localization ◽  
Color Mixing ◽  
Electron Microscopic Localization

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.

scholarly journals Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique.

1986 ◽  
Vol 34 (7) ◽  
pp. 899-907 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Cathepsin B ◽  
Rat Kidney ◽  
Protein A ◽  
Proximal Tubules ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Apical Vesicles ◽  
Collecting Tubules

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.

Sign in / Sign up

Export Citation Format

Share Document