11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
157
6. Incubate slides in RNAse solution (0.1 mg/ml) for 1 hat 37°C.
7. Dehydrate in 70o/o, 90o/o, 100o/o ethanol for 5 min each.
8. Air dry and proceed as described in Chap. 12.
Note: As an alternative to proteinase K treatment, digestion can be performed using pepsin in a concentration of 50-200 1-1g/ml in 0.01 M HCl
Subprotocol 7
Clinical Material Obtained from Solid Tumors: Pretreatment of Slides
with Poly-L-Lysine
The evaluation of interphase nuclei by FISH is especially useful for the cytogenetic analysis of solid tumor material, since in many cases metaphase
chromosomes cannot be prepared. In principle, there are two strategies for
specimen preparation:
• isolation of single cells from solid tumors which are spotted on slides (see
Subprotocol8), and then subjected to in situ hybridization (Hopman et
al. 1988; V an Dekken et al. 1990; Hopman et al. 1991; Arnoldus et al. 1991;
Hopman et al. 1992) or
• direct hybridization to sections of frozen or paraffin-embedded tissues
(Bumset al. 1986; Emmerich et al. 1989b; Waltet al. 1989; Hopman et al.
1991; Arnoldus et al. 1991; Hopman et al. 1992; Bumset al1985; Pringle et
al. 1987; Naoumov et al. 1988).
Only the second strategy provides information about the location of cytogenetically aberrant cells in a histological context. Frazen and paraffin-embedded tissue sections require special pre-treatments that are described in
Subprotocols 9 and 10, respectively.
In general, successful hybridization to material derived from solid tumors depends on efficient permeabilization procedures, which facilitate
probe penetration into cells. For this purpose, proteolytic digestion is
most commonly used. lt should be noted that proteolytic digestion must
be optimized for each sample source. In paraffin-embedded tissue sections
the effect of pepsin digestion can be enhanced by additional treatment with
sodium thiocyanate (Hopman et al. 1992; Hopman et al. 1991). In many
applications tissue sections are mounted on glass slides prior to treatment.
Alternatively, the treatment ( as weil as the hybridization procedure) can be
performed in suspension, followed by mounting. The adhesion of mounted
157
6. Incubate slides in RNAse solution (0.1 mg/ml) for 1 hat 37°C.
7. Dehydrate in 70o/o, 90o/o, 100o/o ethanol for 5 min each.
8. Air dry and proceed as described in Chap. 12.
Note: As an alternative to proteinase K treatment, digestion can be performed using pepsin in a concentration of 50-200 1-1g/ml in 0.01 M HCl
Subprotocol 7
Clinical Material Obtained from Solid Tumors: Pretreatment of Slides
with Poly-L-Lysine
The evaluation of interphase nuclei by FISH is especially useful for the cytogenetic analysis of solid tumor material, since in many cases metaphase
chromosomes cannot be prepared. In principle, there are two strategies for
specimen preparation:
• isolation of single cells from solid tumors which are spotted on slides (see
Subprotocol8), and then subjected to in situ hybridization (Hopman et
al. 1988; V an Dekken et al. 1990; Hopman et al. 1991; Arnoldus et al. 1991;
Hopman et al. 1992) or
• direct hybridization to sections of frozen or paraffin-embedded tissues
(Bumset al. 1986; Emmerich et al. 1989b; Waltet al. 1989; Hopman et al.
1991; Arnoldus et al. 1991; Hopman et al. 1992; Bumset al1985; Pringle et
al. 1987; Naoumov et al. 1988).
Only the second strategy provides information about the location of cytogenetically aberrant cells in a histological context. Frazen and paraffin-embedded tissue sections require special pre-treatments that are described in
Subprotocols 9 and 10, respectively.
In general, successful hybridization to material derived from solid tumors depends on efficient permeabilization procedures, which facilitate
probe penetration into cells. For this purpose, proteolytic digestion is
most commonly used. lt should be noted that proteolytic digestion must
be optimized for each sample source. In paraffin-embedded tissue sections
the effect of pepsin digestion can be enhanced by additional treatment with
sodium thiocyanate (Hopman et al. 1992; Hopman et al. 1991). In many
applications tissue sections are mounted on glass slides prior to treatment.
Alternatively, the treatment ( as weil as the hybridization procedure) can be
performed in suspension, followed by mounting. The adhesion of mounted