Controls:

1.       positive controls: single staining for each of the target proteins using the appropriate label

2.       negative controls: omission of one of the primary antibodies but retaining all of the secondary immunoreagents.

3.       so for a double staining experiment, there are four control conditions:

A.     single immunochemical staining for protein A;

B.      single immunochemical staining for protein B;

C.      multiple immunochemical staining, omitting anti-protein A primary antibody;

D.     multiple immunochemical staining, omitting anti-protein B primary antibody.