There is a lot to say in relation to
antibodies (ab's). What kind of sera should be used; monoclonal or polyclonal?
Which has the advantage?
In general; all ab's can be used that still recognize the epitope after
fixation with the applied fixative. This is one reason that most protocols work
with formaldehyde. This is a mild fixative that can be bought as a stable
solution (formalin). Most of the ab's sold by companies will recognize the epitope after this
fixation (suggestions for
rat). Do you have an
ab producing line yourself
than is testing the only solution. Most of the sold antibodies also work on
acetone fixed cells.
Origin and isotype can be of influence on the staining. And the use of a direct or indirect
staining of the ab is most of the time crucial in detection of the target or not.
Indirect staining of an antibody (using a secondary ab) often leads to a
background staining that ca even can be equal to the specific signal.
a relatively easy intracellular staining of a cytokine like IL-2 in T lymphocytes,
for instance, directly conjugated ab's are available to detect the signal in a
These ab's are available in a large selection and conjugated with different fluorochromes (most of the times FITC, PE or APC).
But when localization of nuclear factors
in the cell should be shown under a fluorescence microscope, an indirect staining
using a secondary ab is preferred. That way the signal is enhanced and makes it
possible to use ab's that
are not widely available as conjugates. The choice of conjugate is depending of
the used (confocal) microscope and the bleaching time of the fluorochrome.