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Multiple labelling using fluorescent labelled secondary antibodies
Common causes of background from secondary antibodies
(If it has been determined that primary antibodies did not contribute to the background)
Improper pre-blocking of the tissues or cells
As a general rule, it is safer to pre-block the tissue with 5% normal serum from the same host species as
the labelled secondary antibody. The IgG in the serum should occupy sticky sites on the tissue or cells to
prevent non-specific binding of the labelled IgG antibody. As mentioned, therefore, with multiple
labelling, secondary antibodies from the same host species should be used.
Never block the tissue with normal serum from the same host species as the primary antibody. For
example, if a primary antibody is made in mouse and normal mouse serum were used for blocking, the
mouse IgG would bind to the sticky sites and be recognised by labelled anti-mouse IgG. A higher
background would result.
Make sure a universal blocker does not contain any IgG that the labelled secondary antibody might cross-
react with. One of the most commonly used protein blockers is bovine serum albumin (BSA). Most of the
commercially available BSA is contaminated with bovine IgG (even some of the higher purity grades),
which might cause problems when a goat primary antibody is used. The labelled secondary anti-goat will
cross-react with bovine IgG significantly since goat and bovine antibodies are somewhat closely related.
Cross-reactivity of the labelled secondary antibodies with the indigenous immunoglobulins
on the tissues or cells
Try to look for a labelled secondary antibody that has been adsorbed against the tissue species. For
example, if the tissue is human tonsil and the primary antibody is made in mouse, use a labelled anti-
mouse IgG that has been adsorbed against human serum proteins so that the anti-mouse IgG will not
cross-react with the indigenous human IgG.
If a mouse tissue that contains immunoglobulins is used and the primary antibody is also made in mouse,
the indigenous immunoglobulins may be blocked with a monovalent Fab fragment of anti-mouse IgG. To
avoid subsequent challenge by the labelled anti-mouse IgG, the tissue may be lightly fixed after Fab
antibody blocking, provided this treatment does not alter the antigen to be detected. Why use an Fab
monovalent antibody? If a divalent antibody is used for blocking, the second binding site on the antibody
might be open to capture a subsequent primary antibody in a multiple labelling protocol and thus amplify
This may be a problem when too many slides are jammed in the same washing chamber.
This may also be a problem when labelling a whole mount or a thick section that allows poor penetration.
If it takes a long time for antibodies to penetrate into the cells, it should also take a long time for the
excess antibodies to come out.
Reactivity of the labelled secondary antibody with the immunoglobulins in the diluent
For example, if a labelled anti-goat antibody is diluted in diluent containing IgG-contaminated BSA, sticky
immune complexes might form due to the cross-reaction of anti-goat with bovine IgG. It is usually not
necessary to dilute a labelled secondary antibody in any protein-containing diluent for a few hours of
incubation. Therefore, it is safer not to add any protein to the antibody diluent to avoid this type of
problem. For a more effective blocking it is better to pre-incubate the tissue with the blocker instead of
adding blocker to the antibody diluent. A blocker containing an IgG, which cross-reacts with the
secondary antibody should not be used even for preblocking.
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Not diluting the secondary antibodies far enough
If background persists after taking all of the above precautions, titrate the labelled secondary antibody
further to obtain a maximal signal to noise ratio. Most of the time the labelled secondary antibodies may
be diluted further than expected.
Possible causes of weak signals from labelled secondary antibodies
(assuming that antigens are present and primary antibodies are working)
There is not sufficient antigen present and an amplification protocol might be needed
Change from a more sensitive detection method to a less sensitive detection method
For example, switching the detection method from immunoperoxidase to immunofluorescence results in a
10 to 100 fold decrease in sensitivity. Sometimes, using a higher concentration of the primary antibody
may solve the problem. Sometimes, an amplification protocol is required to obtain desired signal.
The primary antibody is diluted too far
For example, a working concentration of the primary antibody has been determined previously by
immunoperoxidase to be 0.1 µg/ml. For various reasons, later on, immunofluorescence is tried as another
way of detection on the same antigen using the same concentration of the primary antibody. No signal
could be detected although the fluorophore-labelled secondary antibody was used at the recommended
working dilutions (or at a final working concentration of approximately 2 - 30 µg/ml). Obviously, using a
20 to 300 fold molar excess of the secondary antibody won't help if there is not enough primary
antibodies to begin with.
The secondary antibody does not recognise certain primary antibodies well
Secondary antibodies that are raised against and purified on the solid phase column of immunoglobulins
isolated from "normal serum" may not recognise certain less dominant isotypes of immunoglobulins well.
This is usually not a problem for polyclonal primary antibodies (made in rabbits, goats, guinea pigs etc.)
since polyclonal antibodies usually contain more than one isotype.
A secondary antibody that has been adsorbed against closely related species, such as anti-mouse
adsorbed against rat, only recognises a few epitopes on mouse IgG that are different from rat IgG.
Therefore, if a certain monoclonal mouse IgG primary antibody does not bear a lot of those unique
epitopes, then it may not be recognised well by the labelled secondary antibody.
The secondary antibody cross-reacts with immunoglobulins in the diluent
For example, an anti-goat IgG, diluted in a diluent containing horse serum or BSA contaminated with
bovine IgG, may lose some of its activity due to the cross-reactivity of the antibody with bovine or horse
IgG. The cross-reactivity may also result in higher background due to the formation of sticky immune
Precaution before choosing a mounting medium for Cy2, Cy3, and Cy5 conjugates
Aromatic amines, such as phenylenediamine, can react with cyanine dyes (especially Cy2) and cleave
away half of the cyanine molecule. Use of phenylenediamine as an anti-fading reagent in mounting media
may result in weak and diffused fluorescence after storage of the stained slides. As an alternative,
glycerol-based mounting media containing anti-fading agents such as DABCO or n-propyl gallate may be
used. Organic-based mounting media, such as DPX or methyl salicylate, also may be used for cyanine
dyes. Methyl salicylate remains liquid so the cover slip needs to be sealed with something like nail polish.
DPX will harden into a plastic-like permanent mount. DPX is manufactured by BDH in England.
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