Cover-Glass Embedding in Open-End Capsules for Electron Microscopy

Rosen, Sidney I.

doi:10.3109/10520296209117735

Cited by 22 publications

(4 citation statements)

References 6 publications

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“…Rapid cooling of the embedded specimen after full polymerization facilitated the separation of the cell support from the polymerized resin to a certain extent (Howatson & Almeida, 1958;Brinkley et al, 1967;Flaxman et al, 1969). T o this purpose also various coatings of glass cell supports have been employed, such as carbon (Bloom, 1960;Robbins & Gonatas, 1964), collagen (Heyner, 1963), silicone (Micou et al, 1962;Rosen, 1962), teflon (Chang, 1971) and polytetrafluorethane (Buckley, 1971).…”

Section: R E S U L T S a N D Discussionmentioning

confidence: 99%

A new method for comparative light and electron microscopic studies of individual cells, selected in the living state

Ewijk

Hösli

1975

Journal of Microscopy

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SUMMARY A method is described which permits comparative light and electronmicroscopic studies of cell cultures, cell spreads or single selected cells which have been kept in the Plastic Film Dish (PFD). The PFD is a versatile large surface tissue culture chamber which, for electron microscopy, is mounted with a transparent FEP‐Teflon film bottom. Cells are observed, selected and marked on the PFD‐bottom with a high power inverted light microscope. The cells are fixed and dehydrated with a semi‐automatic device while they are still in situ in the PFD. During the preparation steps for electron microscopy the topographical relationship between individual cells and between cells and cell support is accurately retained. After embedding and polymerization the Teflon film is easily peeled off the polymerized Epon, leaving a replica of the mark around the selected cell. This permits relocation of the selected cells for ultrathin sectioning in a plane plan‐parallel to the original cell support. To enable orientated sectioning of selected cells in a plane perpendicular to the cell support, cells are tagged with Letraset‐letters after original embedding and polymerization. Subsequently the re‐embedded polymerized specimens are orientated in the microtome in a position which permits controlled thin sectioning of the tagged cells in the previously selected plane.

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Section: R E S U L T S a N D Discussionmentioning

confidence: 99%

A new method for comparative light and electron microscopic studies of individual cells, selected in the living state

Ewijk

Hösli

1975

Journal of Microscopy

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“…The principal difficulty with these techniques however, has been the removal of the embedding medium from the glass surface and this problem is most evident where epoxy resins are used. In an attempt to overcome this difficulty various pre-treatments of the glass have been employed, such as silicone (Rosen, 1962;Micou et al, 1962), collagen (Heyner, 1963) and carbon (Robins & Gonatas, 1964).…”

Section: Introductionmentioning

confidence: 99%

A sandwich‐embedding technique for monolayers of cells cultured on Araldite

Smith

Gray

Mackay

1969

Journal of Microscopy

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“…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.…”

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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

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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;

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Cover-Glass Embedding in Open-End Capsules for Electron Microscopy

Cited by 22 publications

References 6 publications

A new method for comparative light and electron microscopic studies of individual cells, selected in the living state

A new method for comparative light and electron microscopic studies of individual cells, selected in the living state

A sandwich‐embedding technique for monolayers of cells cultured on Araldite

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

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