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

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.

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Fluorescence overlay antigen mapping of the epidermal basement membrane zone: I. Geometric errors.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.4.8126380 ◽

1994 ◽

Vol 42 (4) ◽

pp. 555-560 ◽

Cited By ~ 6

Author(s):

S Bruins ◽

M C de Jong ◽

K Heeres ◽

M H Wilkinson ◽

M F Jonkman ◽

...

Keyword(s):

Collagen Type ◽

Basement Membrane Zone ◽

Resolving Power ◽

Geometric Errors ◽

Tissue Sections ◽

Verification Test ◽

Collagen Type Vii ◽

Multicolor Fluorescence ◽

Geometric Verification ◽

Epidermal Basement Membrane

To identify in tissue sections the relative positions of antigen distributions close to the resolving power of the microscope, we have developed the fluorescence overlay antigen mapping (FOAM) procedure. As this technique makes high demands on the geometric fidelity of the overlay image, it is essential to recognize geometric errors resulting from optical imperfections. This applies in particular to the image shift difference (ISD) that may routinely occur during fluorescence overlay. We describe here procedures for assessment and mechanical correction of the ISD in tissue sections. Furthermore, we describe an alignment verification test to assess the accuracy of the ISD correction procedure, using collagen Type VII as the geometric verification marker. These procedures should enable reliable evaluation of relative antigen distributions in tissue sections using photomicrographic multicolor fluorescence overlay. Further details of the FOAM technique, such as color fidelity and its utility for diagnostic and research purposes, will be published separately.

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Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 × 10(3) Mr polypeptide during adhesion complex formation

Journal of Cell Science ◽

10.1242/jcs.96.4.651 ◽

1990 ◽

Vol 96 (4) ◽

pp. 651-660

Author(s):

M.A. Kurpakus ◽

E.L. Stock ◽

J.C. Jones

Keyword(s):

Wound Healing ◽

Basement Membrane ◽

In Vitro Model ◽

Collagen Type ◽

Basement Membrane Zone ◽

Lamina Densa ◽

Vitro Model ◽

Collagen Type Vii ◽

Adhesion Complex

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.

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