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Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a crucial tool in histopathology for detecting proteins in tissues and cells. It uses antibodies to identify the presence and location of specific proteins in tissue samples, making it essential in both diagnosis and research.

Discover how our antibodies are validated for IHC and access the protocols needed for successful experiments.

 

 

Recommended Protocols 

In order to achieve the best results with our antibodies, make sure to use these recommended protocols. The protocols are optimized for our Triple A Polyclonals and PrecisA Monoclonals. Here you will also find our best advice for validation and troubleshooting in IHC.

IHC standard protocol
IHC protocol – Ventana Discovery XT

 

Validation and characterization

Our Triple A Polyclonals and PrecisA Monoclonals antibodies are validated through IHC staining of formalin-fixed, paraffin-embedded (FFPE) tissues in a tissue microarray (TMA) format.

IHC is a qualitative technique, so our skilled team performs thorough manual analysis to ensure antibody specificity across a wide range of human tissues.

For comprehensive protein expression profiling, the antibodies are tested on normal tissues from 144 individuals, representing 44 tissue types, and cancer tissues from 216 patients, including duplicate tumor samples from the 20 most common cancers.

A total of 576 tissue cores are immunostained and analyzed for each antibody. Cancer tissues include both high- and low-grade malignancies. Staining intensity, localization, and the proportion of stained cells are carefully evaluated and manually annotated for each tissue.

 

Enhanced Validation in IHC

To further verify specificity, the validation performed for our antibodies is expanded with application-specific Enhanced Validation. In IHC, two different Enhanced Validation methods may be applied: 

1. Orthogonal validation (verification done with a non-antibody-based method
2. Independent antibody validation (two antibodies targeting the same protein but binding to different parts of the protein thus verifying each other)

Currently, over 5,000 antibodies are validated using at least one of these two enhanced validation methods. Learn more about the methods below. 

 
Watch the animation about Enhanced Validation

 

  • 1. Orthogonal validation compares antibody signal to RNA

    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.

  • 2. Validation by independent antibody

    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.

     

Anti-OTC Antibody
Anti-OTC Antibody

Anti-OTC Antibody

HPA000243
In Stock (10+)
4 311,0 kr
Anti-OSBPL3 Antibody
Anti-OSBPL3 Antibody

Anti-OSBPL3 Antibody

HPA000691
In Stock (10+)
4 570,0 kr
Anti-SPIN3 Antibody
Anti-SPIN3 Antibody

Anti-SPIN3 Antibody

HPA000727
In Stock (10+)
4 570,0 kr
Anti-LAMC1 Antibody
Anti-LAMC1 Antibody

Anti-LAMC1 Antibody

HPA001909
In Stock (10+)
4 311,0 kr
Anti-PDCD6IP Antibody
Anti-PDCD6IP Antibody

Anti-PDCD6IP Antibody

HPA011905
In Stock (10+)
4 311,0 kr
Anti-CCDC90B Antibody
Anti-CCDC90B Antibody

Anti-CCDC90B Antibody

HPA011931
In Stock (10+)
4 570,0 kr
Anti-IBTK Antibody
Anti-IBTK Antibody

Anti-IBTK Antibody

HPA023826
In Stock (10+)
4 570,0 kr
Anti-AFMID Antibody
Anti-AFMID Antibody

Anti-AFMID Antibody

HPA023861
In Stock (10+)
4 570,0 kr
Anti-PAGE4 Antibody
Anti-PAGE4 Antibody

Anti-PAGE4 Antibody

HPA023880
In Stock (10+)
4 311,0 kr
Anti-DIP2C Antibody
Anti-DIP2C Antibody

Anti-DIP2C Antibody

HPA030264
In Stock (10+)
4 570,0 kr
Anti-AKNAD1 Antibody

Anti-AKNAD1 Antibody

HPA030271
In Stock (10+)
4 570,0 kr
Anti-UFD1L Antibody
Anti-UFD1L Antibody

Anti-UFD1L Antibody

HPA030287
In Stock (10+)
4 311,0 kr
Anti-CTGF Antibody
Anti-CTGF Antibody

Anti-CTGF Antibody

HPA031075
In Stock (10+)
3 665,0 kr
Anti-MAML1 Antibody
Anti-MAML1 Antibody

Anti-MAML1 Antibody

HPA037687
In Stock (10+)
4 570,0 kr
Anti-BCS1L Antibody
Anti-BCS1L Antibody

Anti-BCS1L Antibody

HPA037701
In Stock (10+)
4 570,0 kr
Anti-MAT2B Antibody
Anti-MAT2B Antibody

Anti-MAT2B Antibody

HPA037721
In Stock (9)
4 570,0 kr
Anti-ABCA7 Antibody
Anti-ABCA7 Antibody

Anti-ABCA7 Antibody

HPA041564
In Stock (10+)
4 570,0 kr
Anti-KCTD18 Antibody

Anti-KCTD18 Antibody

HPA045498
In Stock (10+)
4 570,0 kr
Anti-NXNL2 Antibody

Anti-NXNL2 Antibody

HPA045526
In Stock (10+)
4 570,0 kr
Anti-RIOK1 Antibody
Anti-RIOK1 Antibody

Anti-RIOK1 Antibody

HPA051446
In Stock (10+)
4 570,0 kr
Anti-ATP5G2 Antibody

Anti-ATP5G2 Antibody

HPA051469
In Stock (10+)
4 311,0 kr
Anti-ZNF513 Antibody
Anti-ZNF513 Antibody

Anti-ZNF513 Antibody

HPA051493
In Stock (10+)
4 570,0 kr
Anti-BAG6 Antibody
Anti-BAG6 Antibody

Anti-BAG6 Antibody

HPA053291
In Stock (10+)
4 311,0 kr
Anti-CFAP97 Antibody
Anti-CFAP97 Antibody

Anti-CFAP97 Antibody

HPA053335
In Stock (10+)
4 570,0 kr
Anti-HBQ1 Antibody
Anti-HBQ1 Antibody

Anti-HBQ1 Antibody

HPA062473
In Stock (10+)
4 570,0 kr
Anti-ZNF781 Antibody
Anti-ZNF781 Antibody

Anti-ZNF781 Antibody

HPA062512
In Stock (10+)
4 570,0 kr
Anti-AGAP2 Antibody
Anti-AGAP2 Antibody

Anti-AGAP2 Antibody

HPA075831
In Stock (10+)
4 570,0 kr
Anti-TDRD6 Antibody
Anti-TDRD6 Antibody

Anti-TDRD6 Antibody

HPA075923
In Stock (10+)
4 570,0 kr
Anti-IL17REL Antibody
Anti-IL17REL Antibody

Anti-IL17REL Antibody

HPA045546
In Stock (10+)
4 570,0 kr
Anti-HSF2BP Antibody
Anti-HSF2BP Antibody

Anti-HSF2BP Antibody

HPA075775
In Stock (10+)
4 570,0 kr