34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
445
- Mastermix for single copy probes:
Mix 5 ml deionized formamide, 2 ml 50% dextran sulfate and 1 ml
20xSSC. Vortex thoroughly, adjust to pH 7.5, fill up to 9 ml with
A. bidest.
- Mastermix for indirect single copy probes plus Cot-l DNA:
Add 250 ~l 100% ethanol to 100 ~l Cot-l DNA (1 ~g/1~l) in a 1.5 ml
Eppendorf tube and spin 30 min at 14,000 rpm in a lab centrifuge. Remove supernatant, add 500 ~l 70% ethanol and vortex. Spin again 15
min at 14,000 rpm. Remove supernatant, dry pellet in a speedvac or
allow to air dry. Add 9 ~l prewarmed (37°C) mastermix for single
copy probes and shake for at least 30 min.
Note: All mastermixes can be aliquoted and stored at -20°C.
For a series of questions to be addressed in a tumor cytogenetic lab by
FISH or FICTION, probes directly labeled with a fluorescent dye or indirectly labeled with biotin or digoxigenin are commercially available
(Q-BIOgene, Vysis, etc.). For the detection of numerical aberrations,
satellite probes specific for centromeric regions of distinct chromosomes
should be applied. Chromosome painting probes can be used for the identification of marker chromosomes and structural chromosome aberrations in metaphase spreads but are generally not recommended for use
in interphase nuclei. For some recurrent translocations, e.g. for the
t(9;22), the t(15;17), or for some deletions, e.g. of the RB gene or the
p53 gene, probes are also commercially available. For other structural
aberrations probe sets have been published which can be obtained
from the scientific community. The latter can be prepared according to
routine protocols and labeled by random primed labeling or nick translation using commercially available kits according to the manufacturers'
instructions (Life Technologies, Eggenstein, FRG; Roche, Mannheim,
FRG). For hybridization, an appropriate amount of labeled probe DNA
should be diluted in the mastermixes described above or in hybridization
buffers supplied by the manufacturers (Schlegelberger et al. 1999).
In this paragraph a series of protocols developed for easy and universal
application of commercial and non-commercial probes are described. For
other commercial probes, follow the recommendations of the supplier.
Probes and probe
preparation
for FISH
445
- Mastermix for single copy probes:
Mix 5 ml deionized formamide, 2 ml 50% dextran sulfate and 1 ml
20xSSC. Vortex thoroughly, adjust to pH 7.5, fill up to 9 ml with
A. bidest.
- Mastermix for indirect single copy probes plus Cot-l DNA:
Add 250 ~l 100% ethanol to 100 ~l Cot-l DNA (1 ~g/1~l) in a 1.5 ml
Eppendorf tube and spin 30 min at 14,000 rpm in a lab centrifuge. Remove supernatant, add 500 ~l 70% ethanol and vortex. Spin again 15
min at 14,000 rpm. Remove supernatant, dry pellet in a speedvac or
allow to air dry. Add 9 ~l prewarmed (37°C) mastermix for single
copy probes and shake for at least 30 min.
Note: All mastermixes can be aliquoted and stored at -20°C.
For a series of questions to be addressed in a tumor cytogenetic lab by
FISH or FICTION, probes directly labeled with a fluorescent dye or indirectly labeled with biotin or digoxigenin are commercially available
(Q-BIOgene, Vysis, etc.). For the detection of numerical aberrations,
satellite probes specific for centromeric regions of distinct chromosomes
should be applied. Chromosome painting probes can be used for the identification of marker chromosomes and structural chromosome aberrations in metaphase spreads but are generally not recommended for use
in interphase nuclei. For some recurrent translocations, e.g. for the
t(9;22), the t(15;17), or for some deletions, e.g. of the RB gene or the
p53 gene, probes are also commercially available. For other structural
aberrations probe sets have been published which can be obtained
from the scientific community. The latter can be prepared according to
routine protocols and labeled by random primed labeling or nick translation using commercially available kits according to the manufacturers'
instructions (Life Technologies, Eggenstein, FRG; Roche, Mannheim,
FRG). For hybridization, an appropriate amount of labeled probe DNA
should be diluted in the mastermixes described above or in hybridization
buffers supplied by the manufacturers (Schlegelberger et al. 1999).
In this paragraph a series of protocols developed for easy and universal
application of commercial and non-commercial probes are described. For
other commercial probes, follow the recommendations of the supplier.
Probes and probe
preparation
for FISH