In situ hybridization of cytospin preparations: a rapid nonisotopic screening method for isolated cells.

A simple and rapid method for light microscopic in situ hybridization on cytospin preparations is described and demonstrated for detection of viral nucleic acid in a virus-infected cell line. Cells were fixed by acetone followed by chloroform, denatured by heat, hybridized at 37 C, and hybridized sites detected with a multiple step procedure (primary anti-biotin antibody, biotinylated second antibody, streptavidin-peroxidase). This method can be used for screening studies at the light microscope level, and offers a useful and simple way to determine optimum hybridization conditions for subsequent electron microscopic investigations.

Postembedding ultrastructural in situ hybridization on ultrathin cryosections and LR white resin sections.

A method was developed for nonisotopic postembedding in situ hybridization (ISH) on ultrathin sections of frozen and of LR White resin-embedded material at the electron microscopic level. The method was successfully applied to detect Epstein-Barr virus (EBV) DNA in the P3HR1 human Burkitt's lymphoma cell line. Each of the steps in the procedure had to be optimized for successful ISH on the frozen and LR White sections.

In situ embedding of cell monolayers cultured on plastic surfaces for electron microscopy.

Various procedures suitable for routine in situ embedding of cell monolayers were tested including: (1) the use of different Epon substitutes, (2) the use of different types of plasticware obtained from different sources, and (3) different methods of preparing capsules for sectioning. Different resins reacted differently with different plastics and type of preparation. Merck Epon substitute bound to most of the plastics tested.

Extraskeletal osteosarcoma with unusual ultrastructural features.

A case of extraskeletal osteosarcoma was observed in the thigh of a 33-year-old male patient. Ultrastructurally the tumor was characterized by the presence of a particular dense type of cell, the nucleus of which showed a characteristic combination of features: large amounts of condensed marginated chromatin, prominent perichromatin granules, vermicellar bodies, and undulating microtubules. The tumor also contained intermediate-type cells with a more typical osteoblastic appearance, and more blastic cells.