Time-resolved fluroescence microscopy: Elimination of autofluorescence in tissue specimens for image cytometry with fluorescent labelled probes
✍ Scribed by Mikko Väisälä; Lahja Sevéus; Ari Kuusisto; Raimo Harju; Erkki Soini
- Publisher
- Elsevier Science
- Year
- 1990
- Tongue
- English
- Weight
- 110 KB
- Volume
- 21
- Category
- Article
- ISSN
- 0739-6260
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✦ Synopsis
This paper describes a rapid and simple method for obtaining epon-embedded specimens for light and electron microscopy from a cell monolayer grown on a culture dish. The cells are washed and fixed in situ with glutaraldehyde, post-fixed with osmium tetroxide, and dehydrated in a series of ethanol. After absolute ethanol, a few drops of epon are poured on the bottom of the dish. The dish is then rolled until a thin layer of epon covers the bottom of the dish. One or two gelatin capsules are filled with epon and placed upside down on one side of the dish. The capsules stand better if they are not completely full of epon. The dish is then let stand at room temperature for 30-60 mm. Epon is polymerized at + 45°Cfor 24 hand at +60°Cfor 24 h. After polymerization, the dish is carefully warmed on a hot plate (+ 100°C) until the epon with the cell layer comes off the bottom of the dish. The gelatin capsules can be used as a handle. The whole monolayer is then stained with toluidine blue in a glass Petri dish, washed in tap water and air dried.
The thinnest area of the epon layer is in the centre of the culture dish. This area can be mounted in DePeX on a glass slide and viewed with a light microscope. Pieces of the rest of the epon layer can be reembedded in different orientations for thin sectioning. In addition, thin sections can be cut from the monolayer adhered to the bottom of the gelatin capsules.