Testing Ab's
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 | Use a sample with cells that are positive for the target
and test these against an other sample (other species, other cell type,
unstimulated for instance) that are negative for the target.
Example: take B lymphocytes as control when you would like to stain T lymphocytes for
IFNy. |
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Block the Ab by incubating it with a 10 fold molar excess of
the target before staining the sample. |
| Add a 10 fold larger amount of the unlabeled from of the specific antibody. All events that occur are due to Fc or fluorochrome binding. |
The second and third one tell something about the specificity of the staining. The
first one about the signal that can be expected. When the antibody is only used
for intracellular staining a control with non permeabilised cells can also be a
good control.
Keep in mind that almost every form of fixation leads to little or more
permeabilisation by itself and loss of epitopes. Fixation of cytospins with acetone
already
permeabilises your cells in a way you can directly stain intracellularly!
Should you have commercial/tested Abs available keep in mind that:
| Some epitopes are more sensitive to fixation than others. No staining
doesn't mean your (fixed) epitope is not there. It just can not be recognized
by the Ab. |
| That not every Ab in conjugated as well as the other. The level of the
signal is not comparable between two Abs. The percentages of positive cells
don't even have to be the same! |
| That Abs can recognize different epitopes. So blocking your sample with one antibody and trying to block the interaction of the other antibody might not work. Double staining (different fluorochromes) for the same target might be the solution. |
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Although we did our best we can
not be held responsible for any mistakes or typing errors. |
