Chapter 33
PROTOCOL
Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
Introduction
A variety of immuncytochemical (Ie) and in situ hybridization (ISH)
methods give insight into the presence or absence of specific proteins
and nucleic acid sequences, or establish kinetic parameters of cells.
The combination of these techniques in the same biological specimen
has proven advantageous in, e.g., simultaneously demonstrating the presence of mRNA and its protein product in a given cell (Dirks et al. 1993;
Trembleau et al. 1993), immunophenotyping cells containing a specific
chromosomal aberration (Gendelman et al. 1985; Porter et al. 1990;
Van den Berg et al. 1991; Weber-Matthiesen et al. 1993), or characterization of cytokinetic parameters of tumor cell populations that are genetically or phenotypically aberrant (Balazs et al. 1991; Van Dekken et al.
1991).
For a successful combination of Ie and non-radioactive ISH, several
factors must be taken into account. Preservation of cell morphology and
protein epitopes, accessibility of nucleic acid targets, cross-reaction between the different detection procedures, and stability of enzyme cytochemical precipitates and fluorochromes all determine the success rate
and sensitivity of the procedure. In a combined approach, it is necessary
to perform immunohistochemistry prior to ISH because several steps in
the ISH technique, e.g. enzymatic digestion, postfixation, denaturation at
high temperatures, and hybridization in formamide, can destroy antigenic
determinants.
Heike Steilen-Gimbel, Saarland University, Department of Neurosurgery, Institute of
Human Genetics, Bldg. 68, Homburg / Saar, 66421, Germany (phone +49-68411626605; fax +49-6841-1626600; e-mail nchstg@med-rz.uni-sb.de)
~ Steffi Urbschat, Saarland University, Institute of Human Genetics, Bldg. 60,
Homburg / Saar, 66421, Germany (phone +49-6841-1626635; fax +49-6841-1626600;
e-mail hgsmur@uniklinik-saarland.de)
PROTOCOL
Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
Introduction
A variety of immuncytochemical (Ie) and in situ hybridization (ISH)
methods give insight into the presence or absence of specific proteins
and nucleic acid sequences, or establish kinetic parameters of cells.
The combination of these techniques in the same biological specimen
has proven advantageous in, e.g., simultaneously demonstrating the presence of mRNA and its protein product in a given cell (Dirks et al. 1993;
Trembleau et al. 1993), immunophenotyping cells containing a specific
chromosomal aberration (Gendelman et al. 1985; Porter et al. 1990;
Van den Berg et al. 1991; Weber-Matthiesen et al. 1993), or characterization of cytokinetic parameters of tumor cell populations that are genetically or phenotypically aberrant (Balazs et al. 1991; Van Dekken et al.
1991).
For a successful combination of Ie and non-radioactive ISH, several
factors must be taken into account. Preservation of cell morphology and
protein epitopes, accessibility of nucleic acid targets, cross-reaction between the different detection procedures, and stability of enzyme cytochemical precipitates and fluorochromes all determine the success rate
and sensitivity of the procedure. In a combined approach, it is necessary
to perform immunohistochemistry prior to ISH because several steps in
the ISH technique, e.g. enzymatic digestion, postfixation, denaturation at
high temperatures, and hybridization in formamide, can destroy antigenic
determinants.
Heike Steilen-Gimbel, Saarland University, Department of Neurosurgery, Institute of
Human Genetics, Bldg. 68, Homburg / Saar, 66421, Germany (phone +49-68411626605; fax +49-6841-1626600; e-mail nchstg@med-rz.uni-sb.de)
~ Steffi Urbschat, Saarland University, Institute of Human Genetics, Bldg. 60,
Homburg / Saar, 66421, Germany (phone +49-6841-1626635; fax +49-6841-1626600;
e-mail hgsmur@uniklinik-saarland.de)