•             

Cart 0

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-TMPRSS2 Antibody
Anti-TMPRSS2 Antibody

Anti-TMPRSS2 Antibody

AMAb91792
In Stock (10+)
4 311,0 kr
Anti-FOXL2 Antibody
Anti-FOXL2 Antibody

Anti-FOXL2 Antibody

AMAb91808
In Stock (10+)
4 311,0 kr
Anti-L1CAM Antibody
Anti-L1CAM Antibody

Anti-L1CAM Antibody

AMAb91830
In Stock (10+)
4 311,0 kr
Anti-CHRDL1 Antibody
Anti-CHRDL1 Antibody

Anti-CHRDL1 Antibody

HPA000250
In Stock (10+)
4 570,0 kr
Anti-SRPRB Antibody
Anti-SRPRB Antibody

Anti-SRPRB Antibody

HPA011173
In Stock (10+)
4 570,0 kr
Anti-CD207 Antibody
Anti-CD207 Antibody

Anti-CD207 Antibody

HPA011216
In Stock (10+)
4 570,0 kr
Anti-TEX13B Antibody

Anti-TEX13B Antibody

HPA011283
In Stock (10+)
4 570,0 kr
Anti-DNAI1 Antibody
Anti-DNAI1 Antibody

Anti-DNAI1 Antibody

HPA021843
In Stock (10+)
4 570,0 kr
Anti-PTPRE Antibody

Anti-PTPRE Antibody

HPA021872
In Stock (10+)
4 570,0 kr
Anti-CRYZ Antibody
Anti-CRYZ Antibody

Anti-CRYZ Antibody

HPA021921
In Stock (8)
4 570,0 kr
Anti-SENP7 Antibody
Anti-SENP7 Antibody

Anti-SENP7 Antibody

HPA027259
In Stock (10+)
4 570,0 kr
Anti-FPR2 Antibody
Anti-FPR2 Antibody

Anti-FPR2 Antibody

HPA029154
In Stock (10+)
4 311,0 kr
Anti-CST9 Antibody

Anti-CST9 Antibody

HPA029190
In Stock (10+)
4 570,0 kr
Anti-PPM1A Antibody
Anti-PPM1A Antibody

Anti-PPM1A Antibody

HPA029209
In Stock (10+)
4 311,0 kr
Anti-TAMM41 Antibody
Anti-TAMM41 Antibody

Anti-TAMM41 Antibody

HPA036834
In Stock (10+)
4 570,0 kr
Anti-CELF2 Antibody
Anti-CELF2 Antibody

Anti-CELF2 Antibody

HPA035813
In Stock (10+)
4 570,0 kr
Anti-NBAS Antibody
Anti-NBAS Antibody

Anti-NBAS Antibody

HPA036817
In Stock (10+)
4 570,0 kr
Anti-BEND4 Antibody
Anti-BEND4 Antibody

Anti-BEND4 Antibody

HPA036850
In Stock (10+)
4 570,0 kr
Anti-SP4 Antibody
Anti-SP4 Antibody

Anti-SP4 Antibody

HPA040395
In Stock (10+)
4 570,0 kr
Anti-OCIAD2 Antibody
Anti-OCIAD2 Antibody

Anti-OCIAD2 Antibody

HPA041090
In Stock (10+)
4 570,0 kr
Anti-GFOD2 Antibody
Anti-GFOD2 Antibody

Anti-GFOD2 Antibody

HPA041112
In Stock (10+)
4 570,0 kr
Anti-TRMT1 Antibody
Anti-TRMT1 Antibody

Anti-TRMT1 Antibody

HPA041130
In Stock (10+)
4 570,0 kr
Anti-PDK3 Antibody
Anti-PDK3 Antibody

Anti-PDK3 Antibody

HPA046583
In Stock (10+)
4 570,0 kr
Anti-GPR139 Antibody

Anti-GPR139 Antibody

HPA046596
In Stock (10+)
4 570,0 kr
Anti-RGS21 Antibody

Anti-RGS21 Antibody

HPA052843
In Stock (10+)
4 570,0 kr
Anti-GLDC Antibody
Anti-GLDC Antibody

Anti-GLDC Antibody

HPA052887
In Stock (10+)
4 570,0 kr
Anti-APBB1IP Antibody
Anti-APBB1IP Antibody

Anti-APBB1IP Antibody

HPA063903
In Stock (10+)
4 570,0 kr
Anti-LYRM2 Antibody
Anti-LYRM2 Antibody

Anti-LYRM2 Antibody

HPA063932
In Stock (10+)
4 570,0 kr
Anti-PNOC Antibody
Anti-PNOC Antibody

Anti-PNOC Antibody

AMAb92015
In Stock (10+)
4 570,0 kr
Anti-A2ML1 Antibody
Anti-A2ML1 Antibody

Anti-A2ML1 Antibody

HPA038847
In Stock (10+)
4 570,0 kr