The possibility of electron microscopic autoradiography of steroids after freeze drying of unfixed testes

Abstract

SUMMARY A technique has been developed for the autoradiographic localization of steroids in testes at the light and possibly electron microscope level. During processing of the tissue there is no contact of tissue with water up to the moment of photographic development of the autoradiographs. Tritium labelled steroids have been introduced into the testis through perfusion of the isolated organ. Small tissue samples were rapidly frozen, freeze dried and fixed with osmium vapour. The fixed tissue was embedded in Epon and thin sections could be cut from this material on an ultra‐cryotome without the aid of floatation liquids. Results with light microscopic autoradiography indicate the potential of this technique. This procedure which avoids any contact of the tissue with water offers good prospects for autoradiography of steroids and other soluble compounds at the electron microscope level. A comparison is made with other proposed techniques for steroid autoradiography.

Validation of Freeze-Drying to Visualize Percutaneous ³H-Estradiol Transport: The Influence of Skin Hydration on the Efficacy of the Method

“…A specially designed desiccator [7,8] was evacuated (about 1 Pa), after which the lid from the sample holder was lifted with a manipulator. Osmium tetroxide (OsO 4 ) vapor was admitted to the working chamber by opening a side port containing 0.1 g solid OsO 4 .…”

Cryotechniques in Biological Electron Microscopy

“…15,this VoL), but the statistics for autoradiography generally require many more sections to obtain reliable data. On a cryoultramicrotome rather smooth thin sections were obtained by sectioning this material at -70°C (Frederik and Klepper 1976), sections which have been employed for the autoradiographic localization at the electron microscope level of steroids in testis (Frederik et al 1977). Thin cryosections from fresh-frozen (no chemical fixation) tissue are the most versatile specimens for studying the localization of diffusible substances by X-ray microanalysis or by autoradiography.…”

Combined quick freezing, freeze‐drying, and embedding tissue at low temperature and in low viscosity resins

“…If the viscosity of the embedding resin is low enough, it is possible to directly infiltrate freeze-dried tissue without diluting the resin with an organic solvent. Several studies have demonstrated the utility of such an approach for the preservation of both ultrastructure and the chemical composition of tissues (Frederik and Klepper, 1976;Ingram, 1980, 1984;McGuffee et al, 1979McGuffee et al, ,1981McGuffee et al, ,1985. Since a primary chemical fixation is a n optional step before freezing (Chiovetti et al, 19861, it is possible to quick-freeze either fixed or unfixed tissue and to avoid all solvent dehydration by freeze-drying.…”