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 (less than 1 mm3) of normal adult skin were rapidly frozen in liquid propane at -190 degrees C and subjected to freeze substitution with 100% methanol at -80 degrees C. Specimens were embedded in Lowicryl K11M at -60 degrees C which was polymerized under ultraviolet radiation at -60 degrees 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.
In this study a variety of immunoelectron microscopic methods were used to define the precise ultrastructural binding site of epidermolysis bullosa acquisita antibodies (EBA-Ab). We used two EBA sera which immunoblotted with the same skin-extracted protein as that labelled by a monoclonal antibody (LH7.2) which is known to react with the carboxy terminus of type VII collagen. Gold-conjugated antibodies were used in two different immunoelectron microscopic procedures to compare the labelling characteristics of EBA-Ab and LH7.2 in normal human skin. Antibody incubations were performed using ultra-thin cryosections of unfixed skin and thin slices of fresh skin (en bloc technique) before conventional fixation and embedding in Epon. Both methods showed similar labelling features for both EBA-Ab and LH7.2. With ultra-thin cryosections there was labelling of the lamina densa and an undefined component of the sublamina densa region. With the en bloc technique, labelling of dermal ends of anchoring fibrils and of amorphous material recently defined as 'anchoring plaques' was evident. There was no labelling of the central banded portions of anchoring fibrils. We conclude that EBA-Ag is localized to the dermal ends of anchoring fibrils in addition to the lamina densa and possibly anchoring plaques, and thus has the same distribution as the carboxy terminus of type VII collagen.
From 4 weeks estimated gestational age (EGA) until the end of the second trimester (24 weeks EGA) the fetal epidermis is covered by a specialised epithelium, the periderm. The origin and function of periderm remain speculative. We have demonstrated, using indirect immunofluorescence and immunoperoxidase staining, that periderm is recognised by a mouse IgM monoclonal antibody (Mab) GB1, which has been raised against a simple extract of human amnion. Immunoelectron microscopy localises GB1 to the amniotic surface of periderm, particularly in association with the microvilli, and also bordering cellular identations of the periderm cells. GB1 antigen (ag) is also expressed by the epithelium of fetal oesophagus, fetal and adult conjunctiva and cornea but is absent in a variety of other fetal and adult tissues including bladder, oral mucosa and thymus. The similar distribution of GB1 ag in both periderm and membranes possibly suggests a common origin and the shared expression with fetal oesophagus and fetal and adult eye may indicate a function related to the fluid environment. We therefore feel that GB1 Mab may be of use in further investigations into the origin, structure and function of human periderm.
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