Comparative study of the techniques for ultrastructural localization of antienzyme antibodies

Wolf D. Kuhlmann and 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 lymph node of several species. Fixation with various glutaraldehyde and formaldehyde fixation procedures were tried. None of those fixatives appeared to inhibit the subsequent antigen-antibody reaction. 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 ┬Ám thickness in a cryostat. The latter method 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.