Case Study: Customized IHC Antibody Optimization Services for Preclinical Research

CASE STUDY

Researchers have adopted the use of immunohistochemistry (IHC) tests for research, prognostic and diagnostic use at an extremely fast rate (1). There are over 3 million commercially available antibodies; however, in a Nature Feature entitled, “Reproducibility crisis: Blame it on the Antibodies”, it was shown that less than 50% of these antibodies can be effectively used for IHC (2). To address this issue, Reveal Biosciences has developed a comprehensive and customizable IHC and immunofluorescence (IF) antibody optimization service to assess the use of specific antibodies in IHC applications and optimize the experimental conditions to achieve the best experimental results.

Reveal offers three levels of antibody optimization (bronze, silver, and gold) depending on your study needs. Antibody optimization services vary from testing a single antibody to assessing multiple antibodies raised against the same biomarker. Additional testing including Western blots and reproducibility is also available.

Here we present a typical BRONZE antibody optimization study to illustrate how this service is performed.

METHOD

Samples

  • Samples may be formalin fixed paraffin embedded (FFPE) or frozen tissue from a wide variety of species depending on the research goals. Control tissues are selected based on known expression profiles determined from published literature or online resources such as the Human Protein Atlas (proteinatlas.org). Reveal can provide or source tissue as needed. Cell pellets with known protein expression can also be used as positive and negative controls.

IHC Procedure (IF procedure available on request)

  1. FFPE tissue samples are sectioned at 4µm onto positively charged slides.
  2. Immunohistochemistry is performed on a Leica Bond automated immunostainer. Heat-induced antigen retrieval (HIER) is performed using either Leica Bond Epitope Retrieval Buffer 1 (citrate buffer, pH 6.0) or Leica Bond Epitope Retrieval Buffer 2 (EDTA buffer, pH 9.0) for 20 minutes.
  3. Primary antibodies are tested on tissue that is known to be positive for the biomarker of interest. Testing is performed using 4 different antibody concentrations under each of the two antigen retrieval conditions listed above.
  4. Novocastra Bond Refine Polymer Detection secondary reagents are used to detect the primary antibodies. Secondary antibodies are visualized using 3,3-Diaminobenzidine (DAB – brown). Hematoxylin (blue) is also applied to serve as a nuclear counterstain.
  5. For negative controls, an isotype control is used instead of the protein-specific primary antibody. Tissue known to be negative for the specific biomarker is also tested if available.
  6. Optimal staining conditions are selected by the experienced IHC scientists at Reveal and reported to the client together with the staining protocol used.
  7. Whole slide images are generated in bright field for the optimal staining conditions selected using a Panoramic SCAN (3D Histech) and are provided to the client.
  8. Once approved by the client, this protocol is available for future IHC experiments.
Figure 1. Representative images of antibody immunostaining on FFPE tumor samples. Positive immunoreactivity was visualized with DAB (brown). Hematoxylin was used as nuclear counterstain (blue). The scale bar represents 50 µm.

RESULTS

Deliverables

Antibody optimization report containing:

  • High resolution snapshots of IHC or IF staining for each antibody tested plus controls
  • Whole slide images of antibody staining under the optimal conditions are provided separately
  • A detailed protocol including optimal staining conditions for each antibody

CONCLUSION

Antibody optimization services facilitate testing and confirmation of which antibodies can be used in IHC and/or IF. Moreover, for each antibody we generate a detailed protocol and quality IHC or IF images are obtained. This customized antibody optimization service allows a comprehensive assessment of whether each antibody of interest can be used for IHC applications and allows protocols to be generated that are applicable to future IHC studies.

REFERENCES

  1. Grand View Research, Immunohistochemistry (IHC) Market Analysis By Product (Antibodies, Reagents, Equipment, Kits), By Application (Diagnostics, Drug Testing), By End-Use (Hospitals, Diagnostic Laboratories), & Segment Forecasts, 2018 – 2025.
  2. Baker, M, Reproducibility crisis: Blame it on the antibodies, Nature. 2015 May 21; 521(7552).