This is to discriminate between non-specific and specific binding. The control antibody is identical in isotype and fluorochrome to the true antibody, it is usually raised against a hapten. As both the detection range of FL-1,2 and 3 and the emission peaks of the various Fluorochromes overlap, compensation is employed when more than one fluorochrome is used. Ths is not necessary when only one antibody or fluorochrome is used. Due to this overlap, signal from FL1 may impinge on FL2, and vice- versa, additionally the same may occur between FL2 and FL3.
Compensation is simply the subtraction of any overlapping signal from one detector to another.
We then check the signal from marker 1 against the signals generated from marker 1 with marker 2. Secondary Staining or Secondary Labelling Quite often an antibody against a marker is not conjugated with a fluorochrome, this handicap can be overcome by using a secondary antibody that is conjugated. Summary The FACScan can detect and record 5 parameters, FSC, SSC, FL1, FL2 and FL3. FL1,2 and 3 detectors measure light fluorescence, which can only be generated by light emission from fluorochromes conjugated to an antibody that is specific to a given surface antigen. To discriminate between a specific signal and non-specificity, i.e. background, we use isotype antibody controls.