Embedding Aid for Coverslip Mounts in Electron Microscopy

Nebel, B. R.; Minick, O. T.

doi:10.1083/jcb.2.4.61

Cited by 7 publications

(1 citation statement)

References 1 publication

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cells were then fixed and dehydrated by simple transfer of the cover slip through the fixation and dehydration reagents. Embedding was then carried out either by placing a drop of the resin on the substrate (6)(7)(8) or by restricting the resin to a certain area with gelatin capsules (10,12,15,16,20) or plastic rings (13). These procedures have suffered from several drawbacks, the principal one being the problem of separating the embedded cells from the surface on which they were grown.…”

mentioning

confidence: 99%

The Use of Millipore Filters in Ultrastructural Studies of Cell Cultures and Viruses

McCombs

Benyésh-Melnick

Brunschwig

1968

The Journal of Cell Biology

View full text Add to dashboard Cite

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 X 10 s virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.One of the drawbacks of the accepted methodology for preparing tissue culture cells for thin sectioning and electron microscopy has been the need for repeated pelleting by centrifugation during the embedding process. Several recent techniques eliminate the necessity for centrifugation, and permit the cultured cells to be studied essentially in their natural state (6-8, 10, 12, 13, 15, 16, 20). These techniques have generally involved growing the cells on a solid substrate such as a glass cover slip. The cells were then fixed and dehydrated by simple transfer of the cover slip through the fixation and dehydration reagents. Embedding was then carried out either by placing a drop of the resin on the substrate (6-8) or by restricting the resin to a certain area with gelatin capsules (10,12,15,16,20) or plastic rings (13). These procedures have suffered from several drawbacks, the principal one being the problem of separating the embedded cells from the surface on which they were grown.The adsorption and growth of tissue culture cells on Millipore filters and the use of these filters as a solid support for the manipulation of the cells during the embedding process, a technique which results in preparations of cells in their natural state, is described in this report. In addition, Millipore filters were used as a support for embedding and sectioning small numbers of virus particles. MATERIALS AND METHODS Cell Cultures and MediaTwo types of cell cultures were employed in this study: fibroblast cultures of serially propagated human embryonic lung, bone marrow, and spleen cells, grown in Petri dishes in a 5% CO 2 atmosphere;

show abstract