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Archival Cytogenetic Slides ERICH GEBHART

Introduction A great number of cytogenetic slides stored worldwide are available for FISH studies by the use of a recovery technique which would allow the use of, e.g., archived tumor material. This permits the application of various FISH techniques - particularly interphase FISH - to reevaluate chromosomal anomalies for summary studies of the diagnosis, prognosis and therapeutic response (Schad, Dewald 1995). A rather uncomplicated technique will be reported which reactivates slides made permanent by embedding in Eukitt (storage age up to 15 years). It is based on the experience obtained from examinations on archival cytogenetic slides prepared from 33 breast carcinomas, from 23 cancerous effusions, from 100 rectal tumors (Gebhart et al1996), and from 65 bone marrow samples of human leukemias.

Outline This technique consists of : A coverslip removal procedure; a pretreatment step for exclusion of vestiges of RNA or proteins, and a modified FISH protocol. The technique can be applied to slides made from the bone marrow of leukemic patients as well as from slides prepared from cell suspensions of human solid tumors and leukemias.

Erich Gebhart, Universitat Erlangen/Niirnberg, Institut fUr Humangenetik, Schwabachanlage 10, Erlangen, 91054, Germany (phone +49-9131-8522351; fax +49-9131-209297; e-mail egebhart@ humgenet.uni-erlangen.de)

B. W. Rautenstrauss et al. (eds.), FISH Technology © Springer-Verlag Berlin Heidelberg 2002

PROTOCOL

142

ERICH GEBHART

Materials Equipment

Buffers and solutions

The equipment for FISH and its microscopic evaluation have been presented in detail in the previous chapters. For the procedures described here in detail, nothing special is required, except for the usual laboratory equipment such as a heating plate, water bath, Coplin jars, etc. -

Xylene (Merck)

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RNase stem solution 10 Ilg RNase A/Ill 2xSCC

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SSC standard saline citrate (0.3 M NaCl +0.03 M Na citrate)

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PBS phosphate buffered saline (8 g NaCI + 0.2 g KCI + 1.15 g Na2H2P04

+ 0.2 g KH 2 P04 )

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Pepsin stock 10% pepsin in a.d.

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Formaldehyde stock: 37% paraformaldehyde in 1x PBS

Procedure Removal of the embedding medium

The first critical step of the procedure is the removal of the coverslips from the archived slides. This will depend on the embedding medium used. The following description refers to embedding media (e.g. "Eukitt") which are soluble in xylene. Slides embedded, for instance, in "Canada balsam" can be recovered much more efficiently than those embedded in "Eukitt" (G. Thiel, personal communication). Slides are put into a Coplin jar which is fIlled with xylene at 37°C and remain therein until the coverslip floats off spontaneously, i. e. 2 to 5 days (depending on the amount of embedding medium to be dissolved). The xylene can be removed daily and replaced with fresh xylene. This daily change of xylene, however, apparently does not generally improve the result, unless the embedding medium has been coated in a rather thick layer. In an example of a stud