Alternatives Antibodies Cell type Difficult cytokines Fluorochromes Other species Protocol References Technique


Origin and type
Secondary Ab
Testing Ab's
Isotype controls
Antibodies to ...


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
human, mouse and 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.
When trying 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 flow cytometer. 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.


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Page was last updated: 05-03-2005