EVALUATION OF IMMUNOELECTRON MICROSCOPIC TECHNIQUES THE STUDY OF BASEMENT-MEMBRANE ANTIGENS

Recent technical advances in immunoelectron microscopy (IEM), includin g methods of pre- and post-embedding IEM and cryoultramicrotomy, have helped to elucidate the precise ultrastructural localization of variou s basement membrane-related molecules. Our objective was to evaluate t he advantages and disadvantages of several different techniques for st udying the ultrastructural organization of basement membrane component s. We found that, while ''on-surface'' immunolabeling of postembedding IEM and cryoultramicrotomy with antitype IV collagen or anti-laminin- 5 antibody clearly demonstrated dense labeling on the lamina densa, pr eembedding IEM with a 1-nm ultra-small gold probe showed labeling only on the epidermal and/or dermal surfaces of the lamina dense, with no specific gold particles being seen within the lamina densa itself. The se results indicate that even ultra-small colloidal gold-labeled antib ody fails to penetrate the lamina densa in preembedding IEM. However, labeling with a GB3 monoclonal antibody against laminin-5 was demonstr able with preembedding IEM and cryoultramicrotomy, but not with postem bedding IEM, probably due to a loss of antigenicity. These results con firm the advantages and limitations of these techniques of IEM and emp hasize the importance of using different techniques of IEM in determin ing the precise ultrastructural distribution of basement membrane anti gens.