Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy

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Abstract
Using low-temperature postembedding techniques for immunoelectron microscopy, we succeeded in demonstrating the precise localization of bullous pemphigoid antigen (BP-Ag) in normal human skin. Small pieces (3) of normal adult skin were rapidly frozen in liquid propane at-190°C and subjected to freeze substitution with 100% methanol at-80°C. Specimens were embedded in Lowicryl K11M at-60°C which was polymerized under ultraviolet radiation at-60°C. Ultrathin sections were incubated with BP sera followed by rabbit anti-human IgG and colloidal-gold conjugated anti-rabbit IgG. Epidermal ultrastructure was generally well preserved: the basal cell plasma membrane and intra- and extracellular components of hemidesmosomes could be resolved. Gold particles were mainly distributed on and around the hemidesmosomes in both intra- and extracellular sites, with most of the labelling being inside the basal keratinocytes and within about 300 nm of the basal plasma membrane. No specific labelling was observed beneath melanocytes or when normal human serum was used as a control instead of BP serum. Our observations indicate that BP-Ag is localized in and around hemidesmosomes in normal human skin and that the antigen has both intracellular and extracellular domains with the major component occurring inside the cells.