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)
- RNase stem solution 10 Ilg RNase A/Ill 2xSCC
- SSC standard saline citrate (0.3 M NaCl +0.03 M Na citrate)
- PBS phosphate buffered saline (8 g NaCI + 0.2 g KCI + 1.15 g Na2H2P04
+ 0.2 g KH 2 P0 4 )
- Pepsin stock 10% pepsin in a.d.
- 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 study on leukemia slides, it could be shown that there
was a considerable difference in the percentage of evaluable slides between
a xylene exposure up to 48 h (40%) and slides exposed 3 to 4 days (10%).
The removal of the coverslips must be controlled several times a day as
a longer direct action of xylene on the nuclei may destroy the latter. The
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)
- RNase stem solution 10 Ilg RNase A/Ill 2xSCC
- SSC standard saline citrate (0.3 M NaCl +0.03 M Na citrate)
- PBS phosphate buffered saline (8 g NaCI + 0.2 g KCI + 1.15 g Na2H2P04
+ 0.2 g KH 2 P0 4 )
- Pepsin stock 10% pepsin in a.d.
- 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 study on leukemia slides, it could be shown that there
was a considerable difference in the percentage of evaluable slides between
a xylene exposure up to 48 h (40%) and slides exposed 3 to 4 days (10%).
The removal of the coverslips must be controlled several times a day as
a longer direct action of xylene on the nuclei may destroy the latter. The