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.

免疫组织化学（IHC）是组织中蛋白质表达空间分析的公认标准。IHC被广泛用于癌症诊断和基础研究。针对具有未知表达模式的蛋白质的新抗体的开发产生了对彻底验证的需求。我们已经应用了人类蛋白质图谱项目和国家癌症研究所抗体门户网站的资源来生成39种癌细胞系和37种正常人类组织类型中12种蛋白质的蛋白质表达数据。将IHC在细胞和组织微阵列的连续切片上使用每种蛋白质的两种独立抗体的结果与原位邻近连接（isPLA）进行了比较，后者需要两种抗体的结合才能产生检测信号。将IHC和isPLA的半定量得分与所有细胞系和组织类型中相应的12个转录本的表达进行比较。我们的结果显示，与IHC相比，mRNA水平与isPLA之间具有更一致的相关性。与IHC相比，isPLA的主要优点包括增加的检测特异性和减少的非特异性染色。我们得出结论，实施isPLA作为IHC的补充，以分析蛋白质表达并在抗体验证管道中进行，可以导致组织中蛋白质的更准确定位。