Orthogonal validation in IHC involves confirming antibody staining using a non-antibody-based method, like RNA-Seq. In IHC, this means comparing the antibody signal to RNA data from the same samples.

For validation, two tissues are selected for each antibody: one with high RNA expression and one with low or no expression of the target protein. The RNA levels in the two tissues should differ by at least five times.

The validation results show both the IHC staining for these two samples and their corresponding RNA data.

 

Example of orthogonal validation in IHC. IHC staining of liver and kidney tissues using the Anti-SLC2A2 polyclonal antibody (HPA028997). The corresponding RNA-Seq data (TPM values) for the same tissues are presentedat the bottom. Liver and colon samples were chosen because of their high and low SLC2A2 RNA expression, respectively.

This validation method involves confirming antibody specificity by comparing at least two antibodies that target the same protein but recognize different epitopes.

If the staining patterns from both antibodies are consistent across a range of relevant tissues, they validate each other. At Atlas Antibodies, 20-44 tissues are tested for each antibody, and four representative tissues are shown for each one.

The validation results include IHC staining images for both antibodies side by side.

Example of validation by independent antibodies in IHC. The two Anti-TCHHL1 antibodies HPA063483 (left, A) and HPA042579 (right, B) target different regions of TCHHL1. The image shows IHC stainings of the two antibodies in the cerebral cortex, kidney, lymph node, and skin tissues. The antibody's staining pattern across positive and negative tissues is comparable, so the two antibodies validate each other.