scholarly journals Localization of lysosomal and peroxisomal enzymes in the specific granules of rat intestinal eosinophil leukocytes revealed by immunoelectron microscopic techniques.

1984 ◽  
Vol 32 (3) ◽  
pp. 267-274 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Quantitative Analysis ◽  
Intestinal Mucosa ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Protein A ◽  
Gold Complex ◽  
Double Labeling ◽  
Specific Granules ◽  
Acyl Coa Oxidase ◽  
Microscopic Techniques

Immunoelectron microscopic localization of lysosomal and peroxisomal enzymes in the eosinophil leukocytes of rat intestinal mucosa was studied by use of rabbit antibodies to the enzymes coupled to protein A-gold complex. Gold particle labeling for the lysosomal enzymes, beta-glucuronidase and cathepsin D, was present on specific granules, with a heavy concentration on their paracrystalline cores. The peroxisomal enzymes, acyl-CoA oxidase and catalase, were also found on these granules. The double labeling procedures using two different combination of anti-acyl-CoA oxidase and anti-beta-glucuronidase or anti-catalase and anti-cathepsin D revealed that these enzymes were simultaneously present in specific granules of the intestinal eosinophils. Quantitative analysis of the labeling on subcellular compartments confirmed that all enzymes examined are significantly localized within specific granules and that there is no significant labeling on other compartments such as the nucleus and cytoplasm. In the control sections incubated with an immunoglobulin G fraction from nonimmunized rabbits, no specific labeling was seen on the granules or other organelles. These findings indicate that enzymes which previously have been identified in some organs as lysosomal and in others as peroxisomal can be found together in eosinophil granules.

scholarly journals Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

1995 ◽  
Vol 43 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D Malide ◽  
N G Seidah ◽  
M Chrétien ◽  
M Bendayan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Endoproteolytic Cleavage

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Immunoelectron Microscopic Study of Glomerular Lesions Using a Postembedding Method with a Protein A-Gold Complex

Nephron ◽  
1987 ◽  
Vol 46 (2) ◽  
pp. 182-187 ◽  
Author(s):  
Mitsuru Nakajima ◽  
Tadaomi Hirota ◽  
Kiyoaki Kusumoto ◽  
Kouji Taira ◽  
Hidekazu Kamitsuji
Keyword(s):  
Microscopic Study ◽  
Protein A ◽  
Gold Complex ◽  
Glomerular Lesions

scholarly journals Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250454
Author(s):  
Lorena Carvelli ◽  
Andrea Carolina Aguilera ◽  
Leila Zyla ◽  
Laura Lucía Pereyra ◽  
Carlos R. Morales ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Complex Formation ◽  
Cathepsin D ◽  
Initial Segment ◽  
Lysosomal Enzymes ◽  
Sperm Maturation ◽  
Principal Cells ◽  
Adult Rat ◽  
Rat Epididymis ◽  
Lysosomal Proteins

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

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.

BETA-GALACTOSIDASE AND BETA-GLUCURONIDASE ACTIVITIES IN INTESTINAL MUCOSA OF INFANTS AND CHILDREN

PEDIATRICS ◽  
1971 ◽  
Vol 47 (4) ◽  
pp. 737-744
Author(s):  
Irena Antonowicz ◽  
Harry Shwachman ◽  
Ishida Sotoo
Keyword(s):  
Celiac Disease ◽  
Intestinal Mucosa ◽  
Lysosomal Enzymes ◽  
Normal Activity ◽  
Acute Stage ◽  
Infants And Children ◽  
Protein Losing Enteropathy ◽  
Per Oral ◽  
Beta Galactosidase ◽  
Duodenal Biopsies

The activity of two galactosidases (lactase and hetero-β-galactosidase) and β-glucuronidase were studied in per oral duodenal biopsies in 50 infants and children. Ten patients served as controls and 40 had nutritional disorders including celiac disease (acute, and in remission), cystic fibrosis (CF), protein losing enteropathy, and some miscellaneous conditions. The values for the 10 control patients expressed in units/gm protein/minute ± S.D. follows: lactase 38.0 ± 13.4, H-β-gal-ase 1.42 ± 0.35., and β-glucuronidase 1.90 ± 0.45. In the acute stage of celiac disease the lactase values were markedly reduced, the H-β-gal-ase normal or slightly reduced, with normal activity for β-glucuronidase. In clinical remission and while still on a gluten-free diet the activity of lactase remained significantly reduced in seven of nine patients even after 2 to 10 years. The lysosomal enzymes H-β-gal-ase and β-glucuronidase were not strikingly affected in patients with CF although four of six patients showed low values for H-β-galactosidase. β-glucuronidase was not affected in a variety of intestinal disorders including those that severely affect the integrity of the intestinal mucosa. In the conditions studied there was no correlation between the activity of the two galactosidases, nor between the two lysosomal enzymes.

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 Double immunocytochemical labeling applying the protein A-gold technique.

1982 ◽  
Vol 30 (1) ◽  
pp. 81-85 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Tissue Section ◽  
Protein A ◽  
Antigenic Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Gold Particles ◽  
Antigenic Sites

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

Pattern Analysis Meets Cell Biology

1999 ◽  
Vol 5 (S2) ◽  
pp. 510-511
Author(s):  
Robert F. Murphy ◽  
Michael V. Boland
Keyword(s):  
Cell Biology ◽  
Fluorescent Dyes ◽  
Protein A ◽  
Chinese Hamster ◽  
Subcellular Location ◽  
Computer Applications ◽  
Double Labeling ◽  
A Cell ◽  
Widespread Availability

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

scholarly journals Electron microscopy of gold-labeled human and equine chromosomes.

1989 ◽  
Vol 37 (9) ◽  
pp. 1443-1447 ◽  
Author(s):  
P E Messier ◽  
R Drouin ◽  
C L Richer
Keyword(s):  
Electron Microscopy ◽  
Chromosome Banding ◽  
Protein A ◽  
Gold Complex ◽  
Chromosome Identification ◽  
Chromosomal Dna ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Antigen Localization ◽  
Chromatin Reorganization

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

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