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Techniques in immuno-electron microscopy. I. Cryosubstitution

G Schaumburg-Lever1, B Fehrenbacher, H Möller

  • 1Universitäts-Hautklinik, Eberhard-Karls Universität, Tübingen, Germany.

Journal of Cutaneous Pathology
|August 1, 1994
PubMed
Summary
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This study optimized cryofixation for skin tissue, finding Lowicryl K4M embedding best for antibody labeling, despite Epon offering superior morphology. Osmium tetroxide addition reduced labeling intensity.

Area of Science:

  • Electron Microscopy
  • Immunohistochemistry
  • Tissue Preservation

Background:

  • Cryofixation is crucial for preserving ultrastructure in biological tissues.
  • Optimizing cryoprotectant mixtures and embedding resins is essential for high-quality immunolabeling.

Purpose of the Study:

  • To evaluate different cryoprotectants, embedding resins, and fixation methods for preserving normal skin ultrastructure and antibody labeling.
  • To determine the optimal method for immunolabeling of skin tissue using monoclonal antibodies against desmoglein and type VII collagen.

Main Methods:

  • Normal skin was cryoprotected with 30% dimethylformamide (DMF) in PBS or RPMI, then frozen in liquid propane.
  • Cryosubstitution at -90°C using methanol with uranyl acetate or osmium tetroxide, followed by embedding in Lowicryl K4M or Epon.

Related Experiment Videos

  • Ultrastructural preservation and antibody labeling intensity (anti-desmoglein, anti-type VII collagen) were assessed.

    • Main Results:

    • Dimethylformamide (DMF) in PBS created an electron-dense precipitate.
    • Epon embedding yielded better overall morphology compared to Lowicryl K4M.
    • Lowicryl K4M embedding provided superior antibody labeling intensity.
    • Osmium tetroxide addition significantly reduced labeling in both Epon and K4M.

    Conclusions:

    • Lowicryl K4M embedding is preferred for immunolabeling of skin tissue after cryofixation, despite potential morphological compromises.
    • Osmium tetroxide should be avoided when high antibody labeling is desired.
    • Optimized cryofixation and embedding protocols are critical for accurate immunohistochemical analysis of skin.