Comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates

Wolf D. Kuhlmann, S. Avrameas, T. Ternynck


Medizinische Universitätsklinik Heidelberg and Institut für Nuklearmedizin,
Deutsches Krebsforschungszentrum Heidelberg, Germany

Unité d’Immunocytochimie, Département de Biologie Moléculaire,
Institut Pasteur Paris, France
Various experimental conditions were tried to stain intracellular IgG globulins within popliteal lymph node cells by the use of specific antibodies and their Fab fragments labelled with horseradish peroxidase (HRP) following two different conjugation procedures. Fixation with 4% formaldehyde/24 hours, 1% formaldehyde and 1% glutaraldehyde/1-1.5 hours and 1-1.5% glutaraldehyde/1-1.5 hours provided satisfactory ultrastructural conservation of the tissues. None of these fixatives appeared to denature IgG globulins. Weak aldehyde fixation may enhance penetration of conjugates; however, ultrastructural detail is poorly preserved and proteins will leak out from insufficiently fixed tissue, thus increasing the possibility of staining artifacts. Best results were obtained by using frozen sections from well-fixed specimens. Conjugates with molar ratios of antibody or Fab fragment to peroxidase being one, seem more effective, although no significant differences were noted between peroxidase conjugates of whole antibody molecules and Fab fragments.