An improved method for processing single cells for electron microscopy utilizing agarose

Authors

  • Lydia C. Yuan,

    1. Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205
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  • Bela J. Gulyas

    1. Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205
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Abstract

An improved method is presented for processing single cells for electron microscopy. Agarose, which has a low (30°C) gelling temperature, was used as an initial embedding medium for single cells (spermatozoa and oocytes) and dissociated cell preparations (luteal cells and spleen cells). Dispersed cells of corpus luteum, spleen, and epididymal spermatozoa were placed in 1.5% agarose after aldehyde fixation. These fixed cells, embedded in agarose, were packed into a dense pellet by centrifugation, postfixed, then embedded in Epon. Mammalian eggs were not centrifuged; instead, they were embedded in agarose discs. Cells embedded in agarose were cooled below 30°C to allow for gelling, then processed for electron microscopy. Because agarose has a low gelling temperature, some heat-labile substances were preserved, as demonstrated by retention of peroxidase activity using the DAB histochemical method. The agarose embedding procedure is both rapid and facile, and has proven to be of value in the handling of fragile single cells for electron microscopic studies.

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