Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells.
Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post-embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze-drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze-drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.
INSERM EPI 99-08, EFS-Alsace, 10 rue Spielmann 67065 Strasbourg, France. daniele.spehner@efs-alsace.fr
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Spehner
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Drillien
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Proamer
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Hanau
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Edelmann
D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology; D004853:Epoxy Resins; D005612:Freeze Drying; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron; D016613:Plastic Embedding / Q000379:methods*
D004853:Epoxy Resins; D000949:Histocompatibility Antigens Class II; C048709:spurr resin