How to Utilize a Denaturing Solution Step to Eliminate Cross-Reactivity
Double or multiple antigen labeling in immunohistochemistry (IHC) classically relies on the existence of primary antibodies raised in different
species or of different IgG isotypes to ensure the specific labeling with the secondary detection systems.1 With an abundance of mouse primary
antibodies and increase of rabbit primary antibodies, it is easier than ever to conduct chromogenic multiplex (IHC) staining. However, not
infrequently, the best-suited antibodies for a study are only available as the same IgG isotype of the same species, and the scarcity of the antigen
makes a direct labeling approach impractical. In order to limit cross-reactivity, an elution step is required.
Biocare’s Denaturing Solution can be used as an elution step when performing a sequential double stain and/or multiplex technique. This
solution “serves to remove previously bound primary and link antibodies leaving only the deposit of chromogen from the previous steps, thus
eliminating any potential for cross-reactivity.”2 The solution is supplied as 2 separate components that are mixed at different dilutions depending
on the antigen retrieval method used (i.e. no retrieval, enzyme digestion, high pH or low pH retrieval solutions).
Get Started: How to Implement Denaturing Solution in Your Protocol
To use, simply deparaffinize tissue and hydrate to water. Digest or retrieve the tissue and block for endogenous peroxidase and non-specific
protein, if desired. Apply the first primary antibody, followed by the appropriate secondary antibody and chromogen (recommended to use the most
“robust” chromogen dyes (such as DAB, AEC, etc.), followed by less “robust’ chromogens (Fast Red, etc.)). Then employ the Denaturing Solution
for 2-5 minutes. After rinsing with buffer, the second primary antibody can be dispensed, followed by the corresponding secondary and chromogen.
With the assurance of no cross reactivity, the slides are ready to be counterstained, dehydrated, and mounted. Let your Rev-‘Elution’ begin!
To learn more about the Biocare’s Denaturing Solution or other dual stain reagents, please contact us anytime at 800-799-9499 or check out the
following links: www.biocare.net and https://biocare.net/product/denaturing-solution-elution-step/
1. Pirici D, et al. Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype J Histochem Cytochem. 2009 Jun; 57(6): 567–575. 2. Handbook:
Immunochemical Staining Methods- 3rd Edition, 1 January 2001, http://www.ihcworld.com/_books/Dako_Handbook.pdf 3. Yuan, W, et al. Immunohistochemical Double and Triple Stain Strategies with CD8, CD103, FOXP3, PD-1
and CK8/18 in Colon Adenocarcinoma: Quantitation, Prognosis and Immunotherapy, March 2016, https://biocare.net/wp-content/uploads/SP-DBLTRPL-100.pdf 4. Tacha, D, et al. Immunohistochemical multiplex staining strategies
with CD8, CD103, PD-1, FOXP3 and pan melanoma cocktail in tumor infiltrating lymphocytes in melanoma, April 2016, https://biocare.net/wp-content/uploads/SP-MPXSTRAT100.pdf