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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-SF3A1 Antibody
Anti-SF3A1 Antibody

Anti-SF3A1 Antibody

HPA000690
In Stock (10+)
4 570,0 kr
Anti-C22orf29 Antibody
Anti-C22orf29 Antibody

Anti-C22orf29 Antibody

HPA000721
In Stock (10+)
4 311,0 kr
Anti-PARN Antibody
Anti-PARN Antibody

Anti-PARN Antibody

HPA006314
In Stock (10+)
4 570,0 kr
Anti-BTN3A3 Antibody
Anti-BTN3A3 Antibody

Anti-BTN3A3 Antibody

HPA011871
In Stock (10+)
4 570,0 kr
Anti-NEGR1 Antibody
Anti-NEGR1 Antibody

Anti-NEGR1 Antibody

HPA011894
In Stock (10+)
4 311,0 kr
Anti-EIF4G2 Antibody
Anti-EIF4G2 Antibody

Anti-EIF4G2 Antibody

HPA016965
In Stock (10+)
4 311,0 kr
Anti-SLC16A8 Antibody
Anti-SLC16A8 Antibody

Anti-SLC16A8 Antibody

HPA075017
In Stock (10+)
4 570,0 kr
Anti-ATP6V1E2 Antibody
Anti-ATP6V1E2 Antibody

Anti-ATP6V1E2 Antibody

HPA075092
In Stock (10+)
4 570,0 kr
Anti-PROSC Antibody
Anti-PROSC Antibody

Anti-PROSC Antibody

HPA023733
In Stock (10+)
4 311,0 kr
Anti-BIN3 Antibody
Anti-BIN3 Antibody

Anti-BIN3 Antibody

HPA023769
In Stock (9)
4 570,0 kr
Anti-MSRA Antibody
Anti-MSRA Antibody

Anti-MSRA Antibody

HPA023804
On demand
4 570,0 kr
Anti-PSMB10 Antibody
Anti-PSMB10 Antibody

Anti-PSMB10 Antibody

HPA030224
In Stock (10+)
4 570,0 kr
Anti-MTO1 Antibody
Anti-MTO1 Antibody

Anti-MTO1 Antibody

HPA030230
In Stock (10+)
4 570,0 kr
Anti-CFAP74 Antibody
Anti-CFAP74 Antibody

Anti-CFAP74 Antibody

HPA030240
In Stock (10+)
4 570,0 kr
Anti-GUCY2C Antibody
Anti-GUCY2C Antibody

Anti-GUCY2C Antibody

HPA037655
In Stock (10+)
4 311,0 kr
Anti-LRCH4 Antibody
Anti-LRCH4 Antibody

Anti-LRCH4 Antibody

HPA037667
In Stock (10+)
4 570,0 kr
Anti-QRICH1 Antibody
Anti-QRICH1 Antibody

Anti-QRICH1 Antibody

HPA037678
In Stock (10+)
4 570,0 kr
Anti-TMEM173 Antibody
Anti-TMEM173 Antibody

Anti-TMEM173 Antibody

HPA038116
In Stock (10+)
4 570,0 kr
Anti-C15orf40 Antibody
Anti-C15orf40 Antibody

Anti-C15orf40 Antibody

HPA042980
In Stock (10+)
4 570,0 kr
Anti-KRTAP3-3 Antibody

Anti-KRTAP3-3 Antibody

HPA042992
In Stock (10+)
4 570,0 kr
Anti-B3GALT4 Antibody

Anti-B3GALT4 Antibody

HPA043001
In Stock (10+)
4 570,0 kr
Anti-EVC2 Antibody

Anti-EVC2 Antibody

HPA048122
In Stock (10+)
4 570,0 kr
Anti-SHOC2 Antibody

Anti-SHOC2 Antibody

HPA048134
In Stock (10+)
4 570,0 kr
Anti-PSMB3 Antibody
Anti-PSMB3 Antibody

Anti-PSMB3 Antibody

HPA048147
In Stock (10+)
4 570,0 kr
Anti-RHEBL1 Antibody

Anti-RHEBL1 Antibody

HPA053111
In Stock (10+)
4 570,0 kr
Anti-RBMY1F Antibody

Anti-RBMY1F Antibody

HPA053147
In Stock (10+)
4 570,0 kr
Anti-BTLA Antibody
Anti-BTLA Antibody

Anti-BTLA Antibody

HPA062029
In Stock (10+)
4 311,0 kr
Anti-BAD Antibody

Anti-BAD Antibody

HPA062105
In Stock (10+)
3 665,0 kr
Anti-LIMD2 Antibody

Anti-LIMD2 Antibody

HPA075172
In Stock (10+)
4 570,0 kr
Anti-CCDC153 Antibody
Anti-CCDC153 Antibody

Anti-CCDC153 Antibody

HPA041497
In Stock (10+)
4 570,0 kr