Dual ISH-IHC Antibody Validation

Bio-Techne continues to undertake rigorous steps to validate antibodies and ensure antibody reproducibility. By combining tissue staining strategies from the Advanced Cell Diagnostics, R&D Systems and Novus Biologicals brands, we are taking a multiomics approach to antibody validation.

Learn about our additional Orthogonal Antibody Validation Using the RNAscope™ ISH Technology to Validate Antibodies for IHC and find Dual ISH-IHC validated antibodies available from R&D Systems.

Additional Orthogonal Antibody Validation Using the RNAscope ISH Technology to Validate Antibodies for IHC

The specificity of protein detection in tissues by immunohistochemistry (IHC) antibodies can be confirmed by detecting RNA transcripts in tissue using RNAscope in situ hybridization (ISH).

ISH-IHC Benefits

  • Morphological context: detection of RNA and protein while preserving the cell and tissue structural organization
  • Similar workflows: from sample fixation, pre-treatment, hybridization, and signal amplification to data analysis
  • Performed sequentially on the same tissue section or separately on serial tissue sections
  • Antibody-independent validation, or "Orthogonal Validation", as proposed by the International Working Group for Antibody Validation (IWGAV) and the five pillars of antibody validation (Uhlen, M. et al. (2016) Nat. Methods 13:823)
  • Growing number of publications utilizing multiomics approach to investigating the regulation of gene expression using Dual RNAscope ISH-IHC.


IHC-ISH Complementary Workflows

IHC-ISH workflow for sample preparation to analysis

CD68/SR-D1 antibody in human tonsil using dual RNAscope ISH and IHC  

CD68/SR-D1 in Human Tonsil Using Dual RNAscope® ISH and IHC. CD68/SR-D1 mRNA (red) and protein (green) was detected in formalin-fixed paraffin-embedded tissue sections of human tonsil probed with ACD RNAScope® Probe (Catalog # 560591) followed by immunohistochemistry using R&D Systems Mouse Anti-Human CD68/SR-D1 Monoclonal Antibody (Catalog# MAB2040) at 5ug/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte HRP Polymer Antibody (R&D Systems, Catalog # VC001). Tissue was stained using ACD RNAscope® 2.5 HD Duplex Detection Reagents (Catalog # 322500). Specific staining was localized to cytoplasm in lymphocytes.


Additional Dual ISH-IHC Resources:

Learn more about RNAscope ISH Assays. Visit our ACD-brand website

Dual RNAscope ISH-IHC webinars and publications.

Download our Dual RNAscope ISH-IHC Application Note


Five Pillars of Antibody Validation

genetic strategic validation Genetic Strategy Validation- Expression of the target protein is compared before and after knockout or knockdown using CRISPR/CAS9 or siRNA/shRNA. If protein expression following knockout or knockdown is substantially reduced, then antibody specificity is ensured. View knockout validated antibodies.
orthogonal validation Orthogonal Validation- The target protein is examined with an antibody independent strategy and compared with results from an antibody-dependent strategy. A correlation between these two strategies indicates specificity between the antibody and its target protein. Examples of antibody independent techniques may include in situ hybridization, quantitative PCR, RNA-seq or mass spectrometry.
independent antibody validation Independent Antibody Validation- The data generated using several antibodies (ideally targeting different epitopes) in the same protein is compared (e.g. molecular weight and cellular localization).  Consistent results imply antibody selectivity to the target protein.
expression of tagged proteins Expression of Tagged Proteins Validation- A tagged protein is used as a standard for comparison in Western blotting and/or immunocytochemistry (ICC). For example, if the distribution of the tagged protein overlaps with the immunofluorescence signal, then antibody specificity is confirmed.
biological strategies validation Biological Strategies Validation- These strategies use defined biological or chemical modulation of protein expression to demonstrate antibody specificity to the target protein. The data is compared across multiple cell lines including positive and negative expressing cells, and multiple species, if applicable.


Additional Antibody Validation Resources:

Download our Development of Validated Monoclonal Antibodies White Paper

Learn more about Bio-Techne’s Genetic Knockout (KO) Antibody Validation

R&D Systems Antibodies: Built for Reproducibility