Immunohistochemistry (IHC) is the accepted standard for spatial analysis of protein expression in tissues. IHC is widely used for cancer diagnostics and in basic research. The development of new antibodies to proteins with unknown expression patterns has created a demand for thorough validation. We have applied resources from the Human Protein Atlas project and the Antibody Portal at National Cancer Institute to generate protein expression data for 12 proteins across 39 cancer cell lines and 37 normal human tissue types. The outcome of IHC on consecutive sections from both cell and tissue microarrays using two independent antibodies for each protein was compared with in situ proximity ligation (isPLA), where binding by both antibodies is required to generate detection signals. Semi-quantitative scores from IHC and isPLA were compared with expression of the corresponding 12 transcripts across all cell lines and tissue types. Our results show a more consistent correlation between mRNA levels and isPLA as compared to IHC. The main benefits of isPLA include increased detection specificity and decreased unspecific staining compared to IHC. We conclude that implementing isPLA as a complement to IHC for analysis of protein expression and in antibody validation pipelines can lead to more accurate localization of proteins in tissue.