US & Canada: 
Email: 
From Guaiacum to HRP - The History of Peroxidase in IHC
Peroxidase enzymes have played a critical role in scientific discoveries for centuries, from early medicinal experiments to their essential use in modern immunohistochemistry (IHC). Among them, horseradish peroxidase (HRP) has become the gold standard for enzymatic labeling in research and diagnostics. But how did HRP earn its place in science, and what led to its widespread use in IHC?
Armoracia Rusticana (Horseradish)
HRP is a peroxidase enzyme derived from the horseradish plant, Armoracia rusticana. Like other peroxidases, HRP catalyzes oxidation reactions using hydrogen peroxide. It is composed of multiple isoenzymes, with HRP C being the most prevalent, consisting of a single polypeptide chain of 308 amino acid residues. Its stability, efficiency, and ability to produce strong colorimetric reactions make HRP an invaluable tool in scientific applications.
HRP From Discovery to Modern Use:
First documented reaction (early 1800s): French chemist Louis Antoine Planche first recorded a peroxidase-catalyzed reaction while analyzing jalap plant resin for medicinal applications. This marked the first known scientific exploration of HRP’s enzymatic abilities.
Ipomoea purga, commonly referred to as Jalap
Oxidation studies (1855): Swiss chemist Christian Friedrich Schönbein discovered the first peroxidase activity in plant and animal tissue by observing the tissues’ ability to oxidize guaiac, a resin derived from Guaiacum tree wood. This study laid the foundation for HRP's use as an oxidative enzyme in scientific research.
Christian Friedrich Schönbein (left) & Guaiacum Officinale (right)
Peroxidase-based blood detection (1864): Dutch scientist Izaac Van Deen discovered that guaiacum could detect blood in feces and urine. The heme in hemoglobin oxidized guaiac acid, producing a distinctive blue color—a principle that later influenced peroxidase-based staining techniques.
Dutch physiologist Izaak van Deen by Jan Ensing
Enzyme-Antibody Conjugation (1966):
Dr. Stratis Avrameas and Dr. Paul Nakane independently developed methods to chemically link HRP to antibodies, enabling enzyme-based antigen detection.This made immunohistochemistry widely accessible, as the brown precipitate could be visualized using a standard light microscope.
Melanoma stained with Bio SB's Melanoma Cocktail Antibody & DAB HRP Brown Chromogen
HRP in IHC:
HRP’s use in IHC revolutionized tissue staining techniques. The HRP-diaminobenzidine (DAB) reaction is now the most widely used enzymatic chromogen reaction in IHC. In the presence of DAB and hydrogen peroxide, HRP produces a brown precipitate, allowing for clear visualization of antibody-antigen interactions with high specificity. This method became popular due to HRP’s stability and efficiency. It provides clear, permanent staining results, which are crucial for pathologists and accurate diagnostics.
While HRP-DAB remains the standard in IHC staining, advancements in chromogen technology have expanded the options available to researchers and clinicians. In addition to DAB, a variety of HRP-based chromogens—such as AEC Red, HRP Green, and HRP Blue—offer alternative visualization methods. Like HRP, alkaline phosphatase (AP) chromogens provide even more flexibility for IHC applications, either as single stains or as complements to HRP chromogens in multiplex stains.
Bio SB's HRP Chromogens