Fluorescence overlay antigen mapping of the epidermal basement membrane zone: III. Topographic staining and effective resolution.

Abstract

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographic staining model in human skin, using structural EBMZ antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and suggests an effective lower limit of some 60-65 nm. Although this limit is not reached under present conditions, our results show that it is possible to distinguish topographic differences of antigen distributions with an upper resolution limit of 200 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topographic reference markers in future applications of the technique aiming at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Preliminary results on this topic are most promising indeed.

Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components

“…For immunofluorescence, the harvested cultures were processed as previously described using the biotin‐streptavidin system 33 . Digital fluorescence microscopy was performed according to Bruins et al 34 allowing detection of low levels of fluorescence.…”

Basement Membrane Reconstruction in Human Skin Equivalents Is Regulated by Fibroblasts and/or Exogenously Activated Keratinocytes

“…Digital fluorescence microscopy was performed according to Bruins (Bruins et al, 1995), allowing detection of low levels of fluorescence.…”

Clinical features and diagnosis of epidermolysis bullosa acquisita

“…BP180, laminin 332, and type VII collagen, the fluorescence overlay antigen mapping (FOAM) technique allows the detection of co-localization of autoantibodies by overlay of the different pictures [128][129][130]. In EBA, labeling of patient's tissue-bound autoantibodies e.g.…”