quality reagents for:
In Situ Hybridization Continued
Detection and Amplification. Factors such as tissue fixation,
endogenous biotin or enzyme activity, desired sensitivity, and
permanency of record should be considered when choosing
either the optimal probe label or subsequent detection system.
Protocols for fluorescence and enzyme based in situ detection
are available in our brochure entitled, “A Guide to Nucleic Acid
Labeling and Detection” (also available online).
Various options are available for fluorescent or chromogenic
visualization of in situ probes. When choosing the appropriate
reagents, it is important to consider:
1. The label on the probe (e.g. biotin, fluorescein, etc.)
2. The visualization method (fluorescence or chromogenic detection)
3. The level of sensitivity required.
Several streptavidin and avidin reagents, as well as antibodies
to labels, are available directly conjugated to the detection
moiety (fluorochrome or enzyme). These “one-step” reagents
are convenient and sensitive enough for many applications.
If additional sensitivity or flexibility is needed, many of the
antibodies to labels can be detected with additional layers
of amplification. For streptavidin- and avidin-based systems,
use Biotinylated Anti-Streptavidin or Biotinylated Anti-Avidin
reagents to amplify signal intensity in FISH applications. For non-biotin/streptavidin or non-biotin/avidin
strategies, affinity-purified secondary antibody conjugates can
be used for signal amplification.
Associated Reagents. Increasing the accessibility of the
labeled probe and detection reagents to the target sequence
enhances sensitivity and specificity of an ISH signal. This
can be achieved by reducing the size of DNA probes and
pretreating tissues with a digestive enzyme. NicKit™ p.s.o.
(MB-1905) has been developed for reducing the
size of DNA probes to an optimal length for ISH without the
problem of over- or under- digestion encountered with other
methods. Proteinase K (VP-Y179) is used to
digest proteins on tissue sections in order to enhance probe
and detection reagent accessibility to the target. This enzyme
unmasks nucleic acids from associated proteins. This step is
often required when tissues have been treated with crosslinking
fixatives like formaldehyde or glutaraldehyde.
A protein blocking reagent is imperative to reduce background
when detecting hybridized ISH probes. 5x In Situ Blocking
Solution (MB-1220) is designed to be used as
a general blocking agent and diluent for fluorescent and