Antibody Control for the Same Species with Block-Between

Design the 2° Antibody Control for the Same Species with Block-Between 

Plan to add controls that confirm successful blocking steps between two sets of antibodies (Table 12.2). Because of the sequential addition of antibodies, the controls are different from other experiments with the indirect method of immunocyto-chemistry. The first 1° antibody is not eliminated because there are no competing antibodies for the first 1° antibody. Also, the no 1° antibody control for the first 1° antibody was done previously when the Dilution Matrix showed it was bound specifically by the 2° antibody. The controls here test the potential binding of the second 1° antibody and second 2° antibody to the first set of antibodies. 

The first control removes the second 1° antibody (Table 12.2), which will show that the second 2° antibody is binding only to the second 1° antibody. The second control reverses the order of the sets of antibodies, so that anti-Ag B is now first and anti-Ag A is second. This will confirm that the second 2° antibody is binding to the correct 1° antibody. 

Another potential problem occurs if the anti-mouse Fab molecules do not block the mouse species-specific sites. Incubation with the mouse anti-Ag A and antimouse 488 fluorophore (Fig. 12.3a) is then blocked with normal mouse serum IgG (Fig. 12.3b, stippled gray). If there is insufficient anti-mouse Fab to block the mouse antibodies from the first set of incubations, then the second 2° antibody will bind to the first set of antibodies. Specifically, the second 1° antibody, mouse anti-Ag B, binds to the Ag B antigen (Fig. 12.3c). Then the lack of anti-mouse Fab fragments 

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