Immunogold Staining Following Freeze Substitution and Low Temperature Embedding after Chemical Fixation or after Cryoimmobilization for Transmission Electron Microscopy (TEM).

INTRODUCTIONIn this method for freeze substitution and low-temperature embedding in resin prior to immunogold staining, lightly fixed tissue pieces are cryoprotected by immersion in polypropylene glycol. The cryoprotected tissues are quench frozen and transferred under liquid nitrogen to vials containing frozen methanol or methanol containing uranyl acetate. The vials are transferred to a substitution vessel where the temperature can be controlled and a nitrogen atmosphere maintained. The temperature is raised (typically at 5°C/h to -90°C) and maintained for 24 h. This temperature is cold enough to prevent recrystallization of water and thus tissue disruption, but high enough for movement of water to occur and allow substitution with the liquid methanol. After 24 h, ~90% of the water has been substituted. The substitution medium is replaced and the temperature is raised to -70°C for 24 h. The substitution medium is changed again and the temperature is raised to -50°C. The tissue is impregnated with Lowicryl HM20 over a period of 1-5 d and the resin is polymerized by UV irradiation. Tissue is then sectioned and stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections, and primary antibodies are visualized by staining with secondary antibodies conjugated to colloidal gold particles. The immunochemically stained sections are contrast stained with uranyl acetate and lead citrate to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). This is the simplest and most versatile of the post-embedding procedures.