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HomeZell-MarkerAngewandte Zellmarkierung → Comparative study of the techniques for ultrastructural localization of antienzyme antibodies




Comparative study of the techniques for ultrastructural localization of antienzyme antibodies

Wolf D. Kuhlmann, H. R. P. Miller

Institut de Recherches Scientifiques sur le Cancer, Villejuif, France



Different methods of fixation and tissue processing were employed to demonstrate intracellular antibody to horseradish peroxidase, Escherichia coli alkaline phosphatase and glucose oxidase in the popliteal lymph node of the rabbit, rat, guinea pig, and mouse. Fixation with 2.5 % glutaraldehyde for 1.5 hours, 1.25% glutaraldehyde and 1% formaldehyde for 1.5 hours and 4% formaldehyde for 24 hours all provided satisfactory ultrastructural conservation of the tissues. None of these fixatives appeared to inhibit the subsequent antigen-antibody reaction. After fixation the tissues were prepared for incubation with antigen in several different ways. Extremely small fragments of lymph node were cut either by hand with a razor blade or by using a tissue chopper and complete cross sections of the node were cut at 40 µ in a cryostat. The 40 µm thick frozen sections gave the most consistently reproducible results in that antibodies to all 3 enzymes were demonstrable, the intracellular penetration of the enzymes was superior with this method, and specific areas in the lymph node could be selected by light microscopy prior to cutting thin sections. Finally, a technique is described whereby antibody antihorseradish peroxidase can be detected in ultrathin frozen sections.

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