Principle of in situ hybridization

Cells in metaphase or interphase are fixed on a
slide and denatured to change the doublestranded
DNA (1 a) into single-stranded DNA
(2). The metaphase or interphase preparation is
then hybridized (3) with DNA sequences that
are complementary to the region of interest and
that have been labeledwith biotin (1 b). The hybridization
site is made visible by means of a
primary antibody against biotin; this antibody
is bound to a fluorochrome (4), e. g., fluorescein
isothiocyanate (FITC). Since the primary signal
is quite weak, a secondary antibody (e. g.,
avidin) bound to biotin is attached (5). A further
primary antibody can then be attached to the
secondary antibody (6). This amplifies the signal,
which can then be demonstrated by bright
fluorescence under the light microscope.