L

Figure 3 Direct (A-C) and indirect (D-H) immunologic labeling techniques used to localize antigens for immunoelectron microscopy. Direct methods. (A) Marker is conjugated to primary antibody (IgG) and (B) to Fab fragment. (C) Renatured hybrid F(ab)2 fragment with dual specificity bridges target antigen and marker. Indirect methods. (D) Marker-conjugated secondary antibody (IgG) binds unlabeled primary antibody (IgG). (E) Marker-conjugated protein A binds unlabeled primary antibody (IgG). (F) Marker-conjugated streptavidin binds biotinylated primary antibody (IgG). (G) Biotinylated secondary antibody (IgG) bridges marker-conju-gated streptavidin and unlabeled primary antibody (IgG). In the unlabeled antibody bridge method (H), unlabeled secondary antibody (IgG) bridges unlabeled tertiary antibody (IgG) to marker and unlabeled primary antibody (IgG) to target antigen; marker is bound by tertiary antibody (antibody to marker). (Reproduced with permission from Gonda MA (1994) Immunochemistry. New York: Marcel Dekker.)

Figure 4 Electron micrograph of natural killer (NK) tumor target (T) cell conjugates 30 min after initial interaction. The post-embedding thin-section immunolabeling method was used to localize an intracellular pore-forming protein believed to have an active role in target cell lysis. Thin sections were labeled with monoclonal antibodies to pore-forming protein and bound antibodies were localized by the indirect immunogold method. The arrows in the NK cells depict the effector granules containing colloldal-gold-labeled pore-forming protein. Scale bar = 1.0 p.m. (Micrograph contributed by J Ortaldo, K Nagashima and MA Gonda.)

Figure 4 Electron micrograph of natural killer (NK) tumor target (T) cell conjugates 30 min after initial interaction. The post-embedding thin-section immunolabeling method was used to localize an intracellular pore-forming protein believed to have an active role in target cell lysis. Thin sections were labeled with monoclonal antibodies to pore-forming protein and bound antibodies were localized by the indirect immunogold method. The arrows in the NK cells depict the effector granules containing colloldal-gold-labeled pore-forming protein. Scale bar = 1.0 p.m. (Micrograph contributed by J Ortaldo, K Nagashima and MA Gonda.)

face detail. Samples may be fixed or unfixed before the labeling. Often it is desirable to fix the sample mildly with dilute solutions of glutaraldehyde (0.1-0.25%) before labeling, especially where the labeling procedure may alter the surface architecture under investigation.

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