Secondary Ab

In some cases it might be useful to stain indirectly. Staining the target first with the specific unlabeled antibody (Ab) and show the presence of this Ab by using a secondary Ab labelled with an enzyme or fluorochrome. Try to avoid using a biotin - streptavidine combination because you probably enhance the background staining.
A fluorochrome has the advantage of the localization at the direct spot of the target. But asks for a special equipped microscope. Preferably with a (digital) camera to ''enhance'' the signal from one channel compared to the signal from another channel. An enzyme has the advantage of being able to use a standard microscope and enhancing the signal by prolonging the colouring step in the protocol. The disadvantage of using an enzyme can be that the staining is not nicely localized and the aspecific staining of (neighbouring) cells can be terribly large. Also enzymes expressed by the cell itself have to be inactivated before being able to do a specific staining.

There are some requirements to the secondary Ab:

	It preferably is directed against an origin not used to detect other targets in the incubation or it is the same as the other specific (primary) Abs. Otherwise a blocking step in between and aspecific blocking Abs in the rest of the incubations are required.
	The Ab has been purified from Abs directed against the Abs of all other species, especially that of the species from the target containing cell.
	The Ab is a Fab fragment which decreases the change of aspecific binding to Fc-receptors in the sample. Some firms even sell preparations that make it possible for you to stain with a third Ab that is of the same origin of the first Ab without aspecific staining.

And in spite of what you now may think often polyclonal sera work best as secondary antibodies. You only have to make sure you use a well prepared and tested preparation.

Although we did our best we can not be held responsible for any mistakes or typing errors.
Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005