11 Formalin-Fixed and Paraffin-Embedded Tissue Sections
149
analysis are not, or no more available, thus giving access to a wide pool of
material hitherto not considered for genetic studies.
Due to distinctive features of the archival material, in situ hybridization in tissue sections is complicated by a variety of problems which arise
either from characteristics of the tissue itself or from preparation procedures. Major difficulties include reduced probe penetration, high level of
tissue autofluorescence, cell overlap or truncated nuclei, and last but not
least, low hybridization efficiency caused by a long-formalin fixation.
To overcome these problems, different pretreatment procedures have
been developed for tissue sections, involving proteolytic digestion (e.g.
treatment with pepsin, trypsin, proteinase K), microwave treatment, preincubation in formamide solutions, acid treatment or the so called chaotropic treatment with NaSCN, first described by Hopman and colleagues
in 1991.
Although light microscopy is well established in histology, in situ hybridization with fluorescence detection has the major advantages (compared
to bright-field microscopic detection), of a higher sensitivity and the possibility of using various fluorescent dyes for multi-target detection. The
application of direct fluorochrome labeled probes greatly facilitates the
detection procedure.
Main diagnostic applications of FISH in tissue sections relate to questions concerning:
- abortion material where fetal and maternal cells may be distinguished,
as in the placenta or where the differentiation between tissue types
allows correlation between genetic markers and morphological features in cases of genetic mosaicism,
- tumor material where tumor cells can be distinguished from normal
cells, and seemingly normal cells can be unmasked to show the presence of genetic abnormalities. Cell clones with different genetic markers can be separated from each other and, because the tissue maintains its original cell formation, can be related directly to the tissue
regions in haemotoxylin and eosin(HE)-stained serial sections.
In the following, we outline a protocol which has proved highly efficient
for hybridization with direct fluorochrome labeled probes on formalinfixed paraffin-embedded tissue sections.
Principles and
applications
149
analysis are not, or no more available, thus giving access to a wide pool of
material hitherto not considered for genetic studies.
Due to distinctive features of the archival material, in situ hybridization in tissue sections is complicated by a variety of problems which arise
either from characteristics of the tissue itself or from preparation procedures. Major difficulties include reduced probe penetration, high level of
tissue autofluorescence, cell overlap or truncated nuclei, and last but not
least, low hybridization efficiency caused by a long-formalin fixation.
To overcome these problems, different pretreatment procedures have
been developed for tissue sections, involving proteolytic digestion (e.g.
treatment with pepsin, trypsin, proteinase K), microwave treatment, preincubation in formamide solutions, acid treatment or the so called chaotropic treatment with NaSCN, first described by Hopman and colleagues
in 1991.
Although light microscopy is well established in histology, in situ hybridization with fluorescence detection has the major advantages (compared
to bright-field microscopic detection), of a higher sensitivity and the possibility of using various fluorescent dyes for multi-target detection. The
application of direct fluorochrome labeled probes greatly facilitates the
detection procedure.
Main diagnostic applications of FISH in tissue sections relate to questions concerning:
- abortion material where fetal and maternal cells may be distinguished,
as in the placenta or where the differentiation between tissue types
allows correlation between genetic markers and morphological features in cases of genetic mosaicism,
- tumor material where tumor cells can be distinguished from normal
cells, and seemingly normal cells can be unmasked to show the presence of genetic abnormalities. Cell clones with different genetic markers can be separated from each other and, because the tissue maintains its original cell formation, can be related directly to the tissue
regions in haemotoxylin and eosin(HE)-stained serial sections.
In the following, we outline a protocol which has proved highly efficient
for hybridization with direct fluorochrome labeled probes on formalinfixed paraffin-embedded tissue sections.
Principles and
applications