scholarly journals Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.

1982 ◽  
Vol 93 (1) ◽  
pp. 223-229 ◽  
Author(s):  
J Roth ◽  
E G Berger
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Protein A ◽  
Rabbit Antibody ◽  
Concerted Action ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Thiamine Pyrophosphatase ◽  
In Trans ◽  
Lowicryl K4m

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.

scholarly journals A mitotic form of the Golgi apparatus in HeLa cells

1987 ◽  
Vol 104 (4) ◽  
pp. 865-874 ◽  
Author(s):  
JM Lucocq ◽  
JG Pryde ◽  
EG Berger ◽  
G Warren
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Dense Matrix ◽  
Tubular Structures ◽  
Serial Sectioning ◽  
Interphase Cells ◽  
Thiamine Pyrophosphatase ◽  
Multiple Copies ◽  
Lowicryl K4m ◽  
Electron Lucent

Galactosyltransferase, a marker for trans-Golgi cisternae in interphase cells, was localized in mitotic HeLa cells embedded in Lowicryl K4M by immunoelectron microscopy. Specific labeling was found only over multivesicular structures that we term Golgi clusters. Unlike Golgi stacks in interphase cells, these clusters lacked elongated cisternae and ordered stacking of their components but did comprise two distinct regions, one containing electron-lucent vesicles and the other, smaller, vesiculo-tubular structures. Labeling for galactosyltransferase was found predominantly over the latter region. Both structures were embedded in a dense matrix that excluded ribosomes and the cluster was often bounded by cisternae of the rough endoplasmic reticulum, sometimes on all sides. Clusters were present at all stages of mitosis examined, which included prometaphase, metaphase, and telophase. They were also identified in conventionally processed mitotic cells and shown to contain another trans-Golgi marker, thiamine pyrophosphatase. Serial sectioning showed that clusters were discrete and globular and multiple copies appeared to be dispersed in the cytoplasm. Their possible role in the division of the Golgi apparatus is discussed.

scholarly journals Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy.

1984 ◽  
Vol 32 (11) ◽  
pp. 1217-1223 ◽  
Author(s):  
L G Altman ◽  
B G Schneider ◽  
D S Papermaster
Keyword(s):  
Room Temperature ◽  
Dimethyl Formamide ◽  
Point Counting ◽  
Rabbit Antibody ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Kidney Microsomes ◽  
Embedding Medium ◽  
Lowicryl K4m ◽  
Accelerated Protocol

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.

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 Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections.

1984 ◽  
Vol 98 (1) ◽  
pp. 358-363 ◽  
Author(s):  
S Fakan ◽  
G Leser ◽  
T E Martin
Keyword(s):  
Protein A ◽  
Nuclear Architecture ◽  
Gold Complex ◽  
Thin Sections ◽  
Border Regions ◽  
Core Proteins ◽  
Interchromatin Granules ◽  
Lowicryl K4m ◽  
Perichromatin Fibrils

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP. The antibodies were localized by means of a protein A-colloidal gold complex. Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies. This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA. It also suggests a possible structural role of some snRNPs in nuclear architecture. Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus.

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.

scholarly journals Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

1991 ◽  
Vol 39 (11) ◽  
pp. 1519-1529 ◽  
Author(s):  
F Laliberté ◽  
M F Laliberté ◽  
I Nonotte ◽  
J P Bali ◽  
C Chevillard
Keyword(s):  
Intracellular Localization ◽  
Protein A ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Thin Sections ◽  
Fundic Mucosa ◽  
Angiotensin I Converting Enzyme ◽  
Fluorescence And Electron Microscopy ◽  
Immunocytochemical Techniques ◽  
Lowicryl K4m

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.

scholarly journals Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

1986 ◽  
Vol 34 (11) ◽  
pp. 1477-1485 ◽  
Author(s):  
K E Loesser ◽  
K J Doane ◽  
F J Wilson ◽  
F J Roisen ◽  
S Malamed
Keyword(s):  
Immunoelectron Microscopy ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Protein A ◽  
Neuronal Cell ◽  
Cytoskeletal Proteins ◽  
Modified Technique ◽  
Adrenocortical Cells ◽  
Thin Sections ◽  
Lowicryl K4m

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

scholarly journals Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.

1990 ◽  
Vol 38 (11) ◽  
pp. 1687-1691 ◽  
Author(s):  
H Shida ◽  
R Ohga
Keyword(s):  
Special Reference ◽  
Immunoelectron Microscopy ◽  
Epoxy Resins ◽  
Protein A ◽  
Epithelial Tissue ◽  
Thin Sections ◽  
Glycoprotein I ◽  
Methacrylate Monomer ◽  
Lowicryl K4m ◽  
Methyl Methacrylate Monomer

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.

scholarly journals GOLGI APPARATUS, GERL, AND LYSOSOMES OF NEURONS IN RAT DORSAL ROOT GANGLIA, STUDIED BY THICK SECTION AND THIN SECTION CYTOCHEMISTRY

1971 ◽  
Vol 50 (3) ◽  
pp. 859-886 ◽  
Author(s):  
Phyllis M. Novikoff ◽  
Alex B. Novikoff ◽  
Nelson Quintana ◽  
Jean-Jacques Hauw
Keyword(s):  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Golgi Apparatus ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Smooth Endoplasmic Reticulum ◽  
Thin Sections ◽  
Golgi Stack ◽  
Golgi Element ◽  
Thiamine Pyrophosphatase

New insights into the ultrastructure and phosphatase localizations of Golgi apparatus and GERL, and into the probable origin of lysosomes in the neurons of fetal dorsal root ganglia and the small neurons of adult ganglia have come from studying thick (0.5–1.0 µ) as well as thin (up to 500 A) sections by conventional electron microscopy. Tilting the thick specimens, by a goniometer stage, has helped to increase our understanding of the three-dimensional aspects of the Golgi apparatus and GERL. One Golgi element, situated at the inner aspect of the Golgi stack, displays thiamine pyrophosphatase and nucleoside diphosphatase activities. This element exhibits regular geometric arrays (hexagons) of interconnected tubules without evidence of a flattened portion (saccule or cisterna). In contrast, GERL shows acid phosphatase activity and possesses small cisternal portions and anastomosing tubules. Lysosomes appear to bud from GERL. Osmium deposits, following prolonged osmication, are found in the outer Golgi element. Serial 0.5-µ and thin sections of thiamine pyrophosphatase-incubated material demonstrate that, in the neurons studied, the Golgi apparatus is a continuous network coursing through the cytoplasm. Serial thick sections of acid phosphatase-incubated tissue suggest that GERL is also a continuous structure throughout the cytoplasm. Tubules of smooth endoplasmic reticulum, possibly part of GERL, extend into the polygonal compartments of the inner Golgi element. The possible physiological significance of a polygonal arrangement of a phosphatase-rich Golgi element in proximity to smooth ER is considered. A tentative diagram of the Golgi stack and associated endoplasmic reticulum in these neurons has been drawn.

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