Hapten-Sandwich Labeling

Wofsy, Leon; Baker, Patricia C.; Thompson, Kristin; Goodman, Joel W.; Kimura, John; Henry, Claudia

doi:10.1084/jem.140.2.523

Cited by 30 publications

(2 citation statements)

References 13 publications

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“…In one experiment, lac-spleen cells were capped with Fl-rabbit antilac followed by lac-ferritin and processed for transmission electron microscopy according to previously described procedures (28) .…”

Section: Labeling Of Cellsmentioning

confidence: 99%

Intramembrane particles and the organization of lymphocyte membrane proteins

Kuby¹,

Wofsy²

1981

The Journal of Cell Biology

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An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP) . Mouse spleen cells were treated with p-diazoniumphenyl-ß-D-lactoside (lac) to modify all exposed cell-surface proteins . Extensive azo-coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen-or antigen-stimulated cultures . When the Lac-modified cell-surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35% decrease (P < 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation . In contrast, there was no significant alteration of P-face IMP densities or distribution . Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens . This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes : (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane .The role of the plasma membrane in the transmission of information can be seen in a number of systems where cells have been shown to be activated by an event at the cell membrane leading to proliferation and/or differentiation (5,8,23,26). Although the mechanism of lymphocyte activation is not known, there is evidence to suggest that the binding of insolubilized antigen or mitogen to membrane receptors may, under some conditions, be sufficient to initiate the cellular activation process (8) .There is considerable evidence for an association between lymphocyte cell-surface proteins and an underlying actinmyosin contractile system (2, 10, 15), which may be critically involved in the transmission of signals to or from the cell interior. However, the role of intramembrane particles (IMP) in this interaction remains unresolved. In erythrocytes, experiments have suggested an interaction of IMP with the underlying peripheral protein, spectrin, as well as with erythrocyte surface proteins (24). In the lymphocyte membrane, associa-THE JOURNAL Of CELL BIOLOGY " VOLUME 88 MARCH 1981 591-598 ©The Rockefeller University Press " 0021-9525/81/03/0591/08 $1 .00 tion...

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Section: Labeling Of Cellsmentioning

confidence: 99%

Intramembrane particles and the organization of lymphocyte membrane proteins

Kuby¹,

Wofsy²

1981

The Journal of Cell Biology

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“…Another method (Truffa-Bachi and Wofsy, 1970;Wofsy et_ a^., 1974) has been used to separate antigen binding cells by means of affinity chromatography. Large polyacrylamide beads were chemically modified with lac (azo-coupled phenyl-3-lactoside).…”

Section: B Biochemical and Physical Differences Between T Cells And mentioning

confidence: 99%

The immune response of tetraparental mice

Schmerr

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Development and application of immunocytochemical staining techniques: A review

Myers¹

1989

Diagnostic Cytopathology

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Since the field of immunocytochemistry was pioneered almost half a century ago, over 30 different immunostaining methods have been developed. Most laboratory investigators, however, are aware of only a few of the more popular techniques. This article, therefore, reviews and places in historical perspective many of the less common methodologies. A discussion of the progression of immunostaining methodology from purely immunologic techniques (employing only antigens, antibodies, and free enzymatic preparations) to procedures that use non-immunologic substances (such as avidin, biotin, and protein A) is presented. Basic principles of immunostaining, including specimen preparation, fixation, control procedures, interpretation, and problems unique to cytopathology, are also discussed. Schematic diagrams that illustrate the nature and arrangement of the immunochemical reagents employed in most of these techniques are provided in order to demonstrate how various materials can be combined to create a desired staining effect.

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Hapten-Sandwich Labeling

Cited by 30 publications

References 13 publications

Intramembrane particles and the organization of lymphocyte membrane proteins

Intramembrane particles and the organization of lymphocyte membrane proteins

The immune response of tetraparental mice

Development and application of immunocytochemical staining techniques: A review

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