Fluorescence and enzyme-based detection reagents from
Vector Laboratories are ideal for in situ hybridization (ISH)
applications, because of their high affinity, high sensitivity, and
Fluorescence ISH. Choosing a detection method frequently
depends on the abundance and accessibility of the target.
Some targets, such as repetitive sequences, can be directly
visualized with a fluorescently labeled probe. See Figure 1.
Less abundant targets may require a method with greater
sensitivity. In these cases, the fluorescent label on the probe
can serve as a tag. Signal amplification is achieved by binding
a biotinylated antibody to this tag followed by a fluorochromelabeled
streptavidin or avidin. See Figure 2.
Additional signal amplification can be achieved using
Biotinylated Anti-Streptavidin or Biotinylated Anti-Avidin
antibodies. These antibodies bind to streptavidin or
avidin, respectively, through their antigen binding sites
and also through the biotin residues. After the application
of the fluorochrome-labeled streptavidin or avidin, the
signal is enhanced by incubation with either Biotinylated
Anti-Streptavidin or Biotinylated Anti-Avidin antibody,
respectively. This step is followed by a second incubation
with fluorochrome-labeled streptavidin or avidin. This
procedure results in the introduction of a greater number of
fluorochromes at the target site. See Figure 3.
Enzyme-based ISH. Probes labeled with biotin,
fluorochrome, or DNP can be detected with an antibody
that is directly conjugated to either peroxidase or alkaline
phosphatase in a “one-step” detection procedure. A wide
choice of substrates is available for these enzyme conjugates. For a significant increase in sensitivity, an
ImmPRESS™ peroxidase polymer, biotin/streptavidin or biotin/
avidin based systems can also be employed for detecting these
Multiple Label ISH and IHC/ISH. Multiple probes with
different labels can be hybridized simultaneously. After
hybridization, the labeled probes can be detected sequentially
using the same strategies employed for single probe detection.
It is also possible to perform ISH and IHC (immunohistochemistry)
in the same tissue section. This is usually done sequentially.
Antigen detection is usually performed first due to possible
loss of antigenicity from the harsh conditions of hybridization.
After localization of the antigen with a precipitating substrate,
the ISH probe is hybridized and detected. The peroxidase
substrates, DAB (SK-4100) or ImmPACT™ DAB (SK-4105), or
the alkaline phosphatase substrates, Vector® Red (SK-5100) or
BCIP/NBT (SK-5400) can be used first for localization of the antigen
because the reaction products of these substrates remain
stable throughout subsequent ISH procedures.
In Situ Hybridization Continued