As compared to most immunohistochemistry (IHC) assays, a minority of IHC tests detect loss of protein expression in tumor tissues. One may encounter unique challenges in assay optimization, validation, and standardization, illustrated here through case studies in implementing Succinate Dehydrogenase subunit B (SDHB) and H3K27me3 IHC assays. For SDHB, antibody protocols showing stronger staining in internal controls and normal tissues resulted in a staining pattern in some cases of SDH-mutated tumors that was difficult to distinguish from retained staining. For H3K27me3, some malignant peripheral nerve sheath tumors (MPNST) and non-MPNST tumors showed heterogeneous staining, with staining results differing between different autostainer platforms used. In IHC tests with loss of protein expression, antibody protocols need to account for optimal staining in internal control elements, in addition to appropriate staining in tumor cells. “Intermediate” staining patterns, such as heterogeneous or weak expression, pose a challenge for protocol calibration and diagnostic interpretation. The choice of tissue used for analytical validation is important and warrants further evaluation and guidance. Establishing an optimal balance between analytical sensitivity and analytical specificity during protocol optimization is key.