1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
9
Chromosomes/DNA fibers
Aspirated or cultured cells
Tissues
Sperm or fertilized eggs
Nucleic acid arrays
Detection
Sample on slide
Denaturation
..
Posthybrid.
wash
probe annealing
to section of DNA
Fig. 2. Schematic illustration of the fluorescence in situ hybridization principle
FISH can be used to analyze a wide variety of cellular materials and each
sample type requires specific pretreatment measures. To preserve morphological detail, when using individual cells or chromosomes obtained
from cell cultures or cells from aspirated clinical samples, a fixation step is
required. Precipitating fixatives such as 70% ethanol and the conventional
fixative used in cytogenetics (methanol:acetic acid; 3:1) are very useful as
they allow cell suspensions to be stored indefinitely at -20°C. Another advantage is that in situ hybridization can often be performed directly on
such fixed cells or with relatively mild proteolytic digestion. Cross linking
fIXatives are sometimes used to improve morphology but it is at the expense of having to use more rigorous proteolysis. Another important
source of material is archival wax embedded tissues which has the advantage that morphology as well as nucleic acids are optimally preserved.
Ad 1: Sample
preparation
(source,
fixation and
unmasking)
9
Chromosomes/DNA fibers
Aspirated or cultured cells
Tissues
Sperm or fertilized eggs
Nucleic acid arrays
Detection
Sample on slide
Denaturation
..
Posthybrid.
wash
probe annealing
to section of DNA
Fig. 2. Schematic illustration of the fluorescence in situ hybridization principle
FISH can be used to analyze a wide variety of cellular materials and each
sample type requires specific pretreatment measures. To preserve morphological detail, when using individual cells or chromosomes obtained
from cell cultures or cells from aspirated clinical samples, a fixation step is
required. Precipitating fixatives such as 70% ethanol and the conventional
fixative used in cytogenetics (methanol:acetic acid; 3:1) are very useful as
they allow cell suspensions to be stored indefinitely at -20°C. Another advantage is that in situ hybridization can often be performed directly on
such fixed cells or with relatively mild proteolytic digestion. Cross linking
fIXatives are sometimes used to improve morphology but it is at the expense of having to use more rigorous proteolysis. Another important
source of material is archival wax embedded tissues which has the advantage that morphology as well as nucleic acids are optimally preserved.
Ad 1: Sample
preparation
(source,
fixation and
unmasking)