Table 1.

Summary of key IHC controls.

Antibody controls
• Provides evidence that the primary antibody is specifically binding to its epitope within a tissue
• Accounts for variability inherent in biological assays
Control type	Example
Negative	Preimmune serum
Negative	Commercial sera from the same species where antibody was produced
Negative	Isotype control
Positive	Independent antibody raised against the same antigen
Tissue controls
• Confirms the validity of the staining pattern within a tissue
• Accounts for variability inherent in biological assays
Control type	Example
Negative	Section of tissue that is known not to express the target antigen
Negative	Samples that are genetically engineered or modified so that they do not express the target antigen
Positive	Section of tissue where the target antigen is known to be expressed
Positive	Section of tissue that contains anatomical structures where the target antigen is known to localize (structure, cell, subcellular localization)
Additional controls
Control Type	Example	Function
Endogenous tissue background	Section of unstained tissue	Detects endogenous signals that could be confused for positive staining
No primary antibody	Section of tissue stained with everything except the primary antibody	Provides evidence that the staining is produced from detection with the primary antibody and not by the detection system or specimen
Absorption	Use of a primary antibody that has been preabsorbed with an excess of antigen	Used to validate antibody specificity (*use in combination with other specificity controls)