Immunogold electron microscopy using skin in michel’s medium intended for immunofluorescence analysis

Abstract

In the investigation of patients with autoimmune blistering skin diseases, immunogold electron microscopy has proved to be a useful approach in identifying target antigens. Such data are often important in establishing accurate subtyping of an individual’s disease and may be relevant to optimizing treatment. Several immunoelectron microscopy techniques have been developed, although many are time-consuming and require sophisticated equipment and trained personnel. In addition, most skin biopsies taken from patients with suspected autoimmune blistering skin diseases received by diagnostic laboratories arrive in transport medium intended for light microscopic immunofluorescence analysis. We have therefore developed a new, quick and reliable direct preembedding immunogold electron microscopy technique that uses, as its substrate, skin sent to the laboratory in Michel’s medium. The approach is illustrated in skin biopsies from patients with bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita. Identification of target antigens in autoimmune blistering skin diseases is helpful in establishing a precise diagnosis. Immunofluorescence analysis, particularly using salt-split skin techniques or dual-labeling confocal microscopy, may provide useful information in helping to classify an individual’s disease, but immunoelectron microscopy (IEM) remains the “gold standard” investigation.1,2 In recent years, colloidal gold has superseded peroxidase as the preferred marker for IEM because of its granular nature and high electron density. It has been used successfully to label extracellular and intracellular antigens in both preand postembedding techniques.3,4 Existing methods, however, are time-consuming and require sophisticated equipment and a high level of technical expertise. Thus, IEM is not always a practical option for most diagnostic immunofluorescence laboratories. Indeed, most skin specimens sent to a diagnostic laboratory will arrive in a transport medium, such as Michel’s medium, which has previously been shown to be ideal for direct immunofluorescence studies.5,6 The aim of our study was to see if we could also use such skin samples as a possible substrate for IEM. In this report we describe a novel preembedding immunoelectron microscopy method that is performed on cryoprotected frozen sections and uses 1 nm colloidal gold in conjunction with silver staining enhancement. To illustrate the technique, we conducted a study to localize the site of antigen-antibody deposition in bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita.