Ferritin‐conjugated protein A A new immunocytochemical reagent for electron microscopy

Templeton, C.L.; Douglas, R. J.

doi:10.1016/0014-5793(78)81256-3

Cited by 20 publications

(4 citation statements)

References 5 publications

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“…The cells were washed three times with 0.1 glycine-buffered saline (pH 8). The washed cells were suspended in PBS, pH 7.4 and incubated for 45 min at room temperature with 75/~l/ml of rabbit antiserum against the sheep transferrin receptor followed by ferritin-protein A as described (23). Note that the ferritin grains are evenly distributed on the cell surface.…”

Section: Early Time Labeling With Ferritin Conjugates and Colloidal Gold Iggmentioning

confidence: 99%

“…Native, washed sheep reticulocytes were incubated with the specific anti-transferrin receptor antiserum at 0*C for 90 rain which resulted in maximum labeling of the cells (5). After washing in PBS, the cells were reincubated at 0*C with ferritinprotein A for 45 rain (23). The unbound antibody and protein A were washed away at 0*C, and the cells were transferred to 37"C and incubated for a further 30 min in culture medium (5).…”

Section: Incubation Of Cells and Fixation Formentioning

confidence: 99%

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Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.

Pan¹,

Teng²,

Wu³

et al. 1985

The Journal of Cell Biology

1,075

741

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Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37°C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5/~m. Inside these large MVEs are round bodies of ~50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nrn bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the ~50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.It is well known that the transferrin receptor is lost during the maturation of the reticulocyte into the erythrocyte (1-4). Recent studies have shown that the maturation process can be followed in vitro (4, 5-7). The loss of the transferrin receptor can be used as a marker of maturation (4-6), the transferrin receptor being released, undegraded, to the medium in vesicular form (6, 7). The transferrin receptor is known to recycle many times during the course of Fe 3÷ delivery without degradation of either the receptor or the natural ligand, transferrin (8-l 1). It has been shown in several systems (8,(12)(13)(14)(15)(16)(17)(18)(19)(20), using either labeled transferrin or antibody against the transferrin receptor, that the ligand and the receptor are internalized into vesicles (endosomes) during incubation at temperatures above 10*C and that this internalization may constitute part of the iron delivery mechanism.Since the transferrin receptor is largely lost from sheep reticulocytes during 24 h of incubation in vitro (5-7), intermediate stages associated with vesicle externalization during receptor elimination might become apparent during the longterm incubation of reticulocytes. No studies to date have addressed the question of the processing that may occur to prepare the receptor for externalization during reticulocyte maturation. We have previously shown (6) that a polyclonal 942 antibody against the transferrin receptor is internalized (as judged by resistance to acid treatment), and that during the course of long-term incubation (several hours at 37"C), this internalized antibody and the surface-bound antibody are lost from the cells along with the antibody-binding capacity of the cells. Since no degraded antibody was found, both internal and surface antibody appear to be eliminated from the cells without degradation.The rate of loss of internalized 125I-antibody as we...

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Section: Early Time Labeling With Ferritin Conjugates and Colloidal Gold Iggmentioning

confidence: 99%

Section: Incubation Of Cells and Fixation Formentioning

confidence: 99%

Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.

Pan¹,

Teng²,

Wu³

et al. 1985

The Journal of Cell Biology

1,075

741

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“…Particulate markers are metal particles conjugated to the final layertsecondary antibody; they are inherently electron dense, so detectable. Ferritin (Templeton et al, 1978) and iron dextran complexes (Geiger et al, 1981) have been used. Ferritin particles are spherical and have an external diameter of around 12 nm, while iron dextran complexes are cylindrical and measure approximately 12 x 24 nm.…”

Section: Dehydration Progression Through Graded Alcoholsmentioning

confidence: 99%

Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia

Sarraf

Alison

Ansari

et al. 1995

Microscopy Res & Technique

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The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF alpha) takes its name from one of its alternative ligands, epidermal growth factor, and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF alpha and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF alpha is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF alpha but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF alpha, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF alpha, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia.

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“…Different technical variations and improvements have been made all along the years in order to enhance the resolution of the results (Singer, 1959;Sainte-Marie, 1962;Sternberger, 1974). More recently, protein A, tagged with different markers, has been introduced in the field of immunocytochemistry for the localization of antigenic sites at the light and electron microscope level (Bachi et al, 1977;Dubois-Dalcq et al, 1977;Romano and Romano, 1977;Roth et al, 1978Roth et al, , 1980Templeton and Douglas, 1978;Miller et al, 1981;Roth, 1982a,b).…”

Section: Introductionmentioning

confidence: 99%

Protein A‐gold electron microscopic immunocytochemistry: Methods, applications, and limitations

Bendayan

1984

J. Elec. Microsc. Tech.

500

321

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The postembedding protein A-gold immunocytochemical approach has been introduced as a n alternative to other techniques for the ultrastructural localization of antigenic sites. The present review deals with the development, the theoretical background, and technical approach of the protein A-gold method as well as the different modifications introduced in order to enhance the resolution of the results and to perform double labelings on the same section. Various examples demonstrate the reliability and the wide range of application of this technique. In addition, some problems, pitfalls, and limitations particular to this method are reported.

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Ferritin‐conjugated protein A A new immunocytochemical reagent for electron microscopy

Cited by 20 publications

References 5 publications

Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.

Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.

Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia

Protein A‐gold electron microscopic immunocytochemistry: Methods, applications, and limitations

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