Quenching involves inactivating endogenous peroxidase enzymes naturally present in tissues (such as in red blood cells, macrophages, or organs like liver, spleen, and kidney). These enzymes can catalyze the chromogenic reaction (e.g., DAB substrate) independently of antibody-antigen binding, causing background staining and false-positive results.
Buffers Used for HRP Quenching
HRP quenching is typically performed by incubating tissue sections in a buffer containing hydrogen peroxide (H₂O₂), an oxidizing agent that irreversibly inactivates endogenous peroxidases.
- Common Buffer: 3% hydrogen peroxide solution diluted in distilled water or buffered saline (PBS or TBS). The pH is generally neutral (around 7.0–7.4) to preserve tissue morphology.
- Alternatives: Some protocols use hydrogen peroxide diluted in methanol (e.g., 0.3% H₂O₂ in methanol) for enhanced penetration and efficiency, especially on frozen sections.
Typical HRP Quenching Protocol
- After deparaffinization and antigen retrieval, rinse slides in PBS or TBS.
- Incubate sections in 3% hydrogen peroxide solution for 10–15 minutes at room temperature.
- Rinse thoroughly in PBS or TBS to remove residual peroxide.
- Proceed with blocking nonspecific sites and antibody incubation steps.
Importance of Buffer Choice in Quenching
The buffer used to dilute hydrogen peroxide helps maintain pH stability and tissue integrity. The most commonly used buffers are:
- PBS (Phosphate Buffered Saline): Physiological buffer compatible with most IHC protocols.
- TBS (Tris Buffered Saline): Slightly more alkaline, sometimes preferred depending on antibody or detection system.