Two different types of negative quality controls are 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 suggest the best tissue to use as a positive control and indicate a tissue for a negative control.
Negative reagent control
The negative reagent control aims to ensure the 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 like the positive control. Still, the primary antibody is omitted and replaced with another solution, such as isotype, tissue culture media, buffer, or dilute non-immune serum of the same species as the primary antibody. It is essential to note that an antibody diluent is not an acceptable negative reagent control.
Negative tissue control
A negative tissue control should be tissue that does not contain the targeted antigen to ensure no 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, where specific tissue components should permanently stain negative.
