Table 1. . Steps of antibody validation for immunohistochemistry, integrating some of the pillars from Uhlen et al.

Steps of antibody validation	Summary	Common pitfalls	Significance
Step 1. Architectural or subcellular localization of expression	Illustration of the expected biologic localization of signal for the target of interest Includes tissue type, histologic subtype, cellular and subcellular compartments	Nonspecific staining patterns due to suboptimal antibody concentration	Provides early proof of antibody specificity Justifies further validation efforts
Step 2. Antibody optimization	Optimization of assay conditions Includes optimization of antigen retrieval buffer, antibody concentration and incubation conditions	Proper assay conditions often not indicated by the vendor	Ensures that all subsequent validation steps will be conducted under optimal assay conditions
Step 3. Uhlen pillar 2 Orthogonal methods of validation and/or	Utilization of independent methods to prove antibody specificity. Includes western blot, mass spectrometry etc.	Positive band in a western blot for nonspecific antibodies; multiple bands in a western blot for specific antibodies	Provides additional proof of antibody specificity by one or more independent methods
Step 3. Uhlen pillar 1 Genetic methods of validation and/or	Genetic manipulation of the expression of the target of interest to generate positive and/or negative controls	Overexpression or underexpression of the target of interest in wild-type cell lines	Links the genetic basis of the target of interest with the corresponding protein product
Step 3. Uhlen pillar 3 Independent epitope validation	Correlation of multiple antibodies with nonoverlapping epitopes for the target of interest	Identification of a second antibody for the target of interest	Provides substantial proof for the specificity of both antibodies
Step 4. Antibody reproducibility	Demonstration of antibody sensitivity and specificity across different runs, operators, manual vs automated staining methods and lots	Inherent heterogeneity of the target of interest; vendor lot variability	Proves that assay is robust and ready to use