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First of all we have to distinguish to monoclonal from the polyclonal sera.
Polyclonal sera seem to have a higher (aspecific) background than monoclonals.
Depending on the fact whether the serum is affinity purified or not. When you would like to detect more epitopes (from one molecule) to enhance your
signal it might sometimes be better to use different monoclonals in a cocktail. And to make
sure, for instance in the case of cytokines, you don't detect receptor bound
proteins you should use a (target)blocking monoclonal ab. Shouldn't you have
such an ab available you can preincubate fixed cells with the unconjugated ab
before permeabilization and staining with the conjugated Ab. That way the signal
due to receptor bound protein will be limited to a minimum.
All Abs have to be titrated before use. Suboptimal concentrations often lead to
suboptimal signals. This may look obvious but a lot of people don't take notice
of this. It means that the amount of cells per sample should be comparable.
Lower numbers of target cells are not a problem. It is the increasing number of
expected positive cells you should take care of. Most concentrations of Ab will
be between 0.5 and 5 µg/ml, some vendors even get nice specific staining with
concentrations
between 0.5 and 0.15 µg per test. There is a difference between companies when
they state mass. It might be the total mass of protein (antibody and
fluorochrome) or only the amount of antibody.
To prevent increasing backgrounds we prefer to work with directly conjugated
Abs. At the other hand unconjugated abs make you more flexible in making
combinations with other (conjugated) abs. And using a secondary ab can increase
you signal. On the other hand it easier to do multiple colour staining when the
abs
are directly conjugated. Some people prefer doing sequential staining when they
do multiple colour staining, but we didn't see any real differences when incubating all
directly conjugated abs together.
What we did see is that mouse abs seem to give a lower background than rat abs in
a cytokine staining. And FITC conjugated abs give lower (no) background compared
to
PE labelled abs when B cells are present in the sample. Goat and rabbit
(polyclonal) abs don't
seem to give any problems. These are tested in the cyto-spins to look at
localisation of proteins. For all abs it is crucial that the company is doing a
thorough purification and quality check.
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Origin and type
