There are two different types of negative controls used in IHC, and it is BEST practice to use both. One is a negative reagent control, and the other is a known negative tissue control. Reviewing the primary antibody product insert sheet may provide suggestions not only on what is the best tissue to use as a positive control, but it may also give you an indication of a tissue to use for a negative control, as well.
Negative reagent control:
The purpose of the negative reagent control is to ensure specificity and sensitivity of your primary antibody and rule out additional/non-specific staining when the primary antibody is removed from the protocol. When using a negative reagent control, a section of patient tissue is stained in the same manner as the positive control, but the primary antibody is omitted and replaced with some other solution, such as isotype, tissue culture media, buffer, or dilute non-immune serum of the same species as the primary antibody It is important to note that an antibody diluent by itself is not an acceptable negative reagent control.
Negative tissue control:
A negative tissue control should be tissue that does
NOT contain the targeted antigen, in order to ensure there is not positive staining which may indicate nonspecific background staining. A known negative staining tissue may be placed on the same slide as the positive control. This tissue should receive all the same preparation and staining steps as the patient tissue. This also may be an internal control in the positive control tissue itself, where certain components of the tissue should always stain negative.
