The outcome of an experiment performed under the experimenter's conditions cannot be definitely known and so the results must be interpreted relative to controlled conditions - so far as the experimenter can control them anyway. For instance, if one were studying drug induced protein X translocation in a monolayer epithelial culture using fluorescence immunohistochemical techniques to visualize the translocation of the protein in the culture, then it would not be sufficient enough to base one's conclusions on just observing the results on a culture treated with the drug. At the level of drug effect one should at least use a control culture that was not exposed to the drug; and as a further control this group should be exposed to the vehicle used to dissolve the drug. Then you may be able to draw some conclusions about the drug effect on protein translocation.

At the immunohistochemical level you have to determine several things about your experimental setup under controlled conditions before you can confidently draw your conclusions about the results you have observed. Broadly, you can classify these conditions as positive controls and negative controls; controls that will give you a frame work to establish the specificity of your antibody. But before you even consider such controls make sure your imaging system has been optimized for the experiment at hand. For example is the filter set you are using optimal for the fluorophore you have employed? In your fluorescence immunohistochemical experiment you may not see the expression of protein X because you may have had the incorrect filters in place. Are you using the minimal necessary exitation intensity? Too much intensity can create false positive signals.

How do you know the antibody you have used can detect your protein? As a positive control use a cell line that is definitely known to express this protein. If you are doing your experiment in monolayer cultures then perform the positive control in a monolayer culture. If you are doing the experiment in tissue slices then perform the positive control in a tissue type slice known to express this protein. The point is to control variability in the system by doing your control experiments in similar experimental conditions. As a negative control you can use a cell line that does not express the protein. Or better yet preincubate the antibody with the peptide against which it was raised.

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