34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
447
Hybridization mixture for indirectly labeled painting probes (Oncor, AGS):
Painting probes can be applied directly or can be handled as described for
commercial single-copy probes. In general, predigestion of the slides is
recommended.
Hybridization mixture for directly labeled probes (Vysis):
Directly labeled probes should be premixed in the supplied buffers according to the manufacturers' instructions. Alternatively, or for the combination with indirectly labeled probes, dilution in the mastermixes described
for indirectly labeled centromeric or single copy probes is possible.
Procedure
Slide preparation for FISH
Preparing high quality slides is one of the most critical steps for successfully performing FISH. In general, cytogenetic preparations as described
above, conventional unstained peripheral blood or bone marrow smears,
touch preparations and cell suspensions from fresh and frozen tissue are
applicable for FISH. The use of formalin-fIXed paraffin-embedded tissue,
tissue sections and stained blood or bone marrow smears is not recommended for routine use and should be restricted to labs experienced with
the technical considerations of these techniques. In order to facilitate the
correlation of the hybridization signals to individual nuclei, cells should
not be packed too densely. The number of cells per cytospin slide should
not exceed 3,000 cells (diameter of the area containing cells = 0.5 em). In
imprints and smears cells often overlap, but there are usually areas in the
periphery where the cells lie separated from each other. Only such areas
should be evaluated. The erythrocytes should lie flat on the slide and not
be standing on end. Sometimes, this is only possible at the end of the
smears. Prior to hybridization the quality of slides should be checked
by phase contrast microscopy. Areas qualifying for hybridization are characterized by a high density of clearly separated grayish nuclei lacking any
surrounding cytoplasm and should be marked on the back of the slide
with a diamond pencil. In general, all specimens can be hybridized without
any pretreatment. Nevertheless, in order to enhance the hybridization efficiency, predigestion of the slides according to the following or similar
protocols is recommended for most tumor specimens.
447
Hybridization mixture for indirectly labeled painting probes (Oncor, AGS):
Painting probes can be applied directly or can be handled as described for
commercial single-copy probes. In general, predigestion of the slides is
recommended.
Hybridization mixture for directly labeled probes (Vysis):
Directly labeled probes should be premixed in the supplied buffers according to the manufacturers' instructions. Alternatively, or for the combination with indirectly labeled probes, dilution in the mastermixes described
for indirectly labeled centromeric or single copy probes is possible.
Procedure
Slide preparation for FISH
Preparing high quality slides is one of the most critical steps for successfully performing FISH. In general, cytogenetic preparations as described
above, conventional unstained peripheral blood or bone marrow smears,
touch preparations and cell suspensions from fresh and frozen tissue are
applicable for FISH. The use of formalin-fIXed paraffin-embedded tissue,
tissue sections and stained blood or bone marrow smears is not recommended for routine use and should be restricted to labs experienced with
the technical considerations of these techniques. In order to facilitate the
correlation of the hybridization signals to individual nuclei, cells should
not be packed too densely. The number of cells per cytospin slide should
not exceed 3,000 cells (diameter of the area containing cells = 0.5 em). In
imprints and smears cells often overlap, but there are usually areas in the
periphery where the cells lie separated from each other. Only such areas
should be evaluated. The erythrocytes should lie flat on the slide and not
be standing on end. Sometimes, this is only possible at the end of the
smears. Prior to hybridization the quality of slides should be checked
by phase contrast microscopy. Areas qualifying for hybridization are characterized by a high density of clearly separated grayish nuclei lacking any
surrounding cytoplasm and should be marked on the back of the slide
with a diamond pencil. In general, all specimens can be hybridized without
any pretreatment. Nevertheless, in order to enhance the hybridization efficiency, predigestion of the slides according to the following or similar
protocols is recommended for most tumor specimens.