Antigen retrieval is an important step in immunohistochemistry (IHC) because it helps to unmask or expose epitopes on antigens that are normally masked or hidden. The process involves applying heat, pH change, or proteolytic digestion to the tissue section to disrupt fixation-induced cross-linking of proteins and reveal antigenic sites. This improves the specificity and sensitivity of antibody staining in IHC and can help to avoid false negative results.
Fixation can cause cross-linking of proteins and other molecular changes that can obscure the antigenic epitopes. Antigen retrieval techniques can help to reverse these changes and expose the antigens for antibody binding.
Antigen retrieval is a process used in immunohistochemistry (IHC) to enhance the detection of antigens on tissue sections by improving their accessibility to antibodies. There are several methods of antigen retrieval, including:
Heat-induced epitope retrieval (HIER): This method involves boiling the tissue sections in a buffer solution to denature the proteins and expose the antigens.
Benefits: HIER is a simple and widely used method that can be easily performed in most laboratories.
Downsides: HIER can cause tissue damage and artifactual changes, leading to inconsistent results.
The choice of buffer for HIER can vary depending on the type of tissue being analyzed and the specific requirements of the IHC experiment. Some commonly used buffers for HIER include:
- Tris-EDTA (TE) buffer: This buffer has a neutral pH and is often used for retrieval of antigens in acidic tissues such as muscle or nerve.
- Tris-HCl buffer: This buffer has a neutral pH and is often used for retrieval of antigens in basic tissues such as liver or spleen.
- Citrate buffer: This buffer has an acidic pH and is often used for retrieval of antigens in formalin-fixed paraffin-embedded (FFPE) tissues.
- Ethylenediaminetetraacetic acid (EDTA) buffer: This buffer has a neutral pH and is often used for retrieval of antigens in tissues containing calcium such as bone or teeth.
Enzyme-based retrieval: This method involves using proteolytic enzymes to digest the tissue and expose the antigens.
Benefits: Enzyme-based retrieval is more gentle than HIER and can result in less tissue damage.
Downsides: The choice of enzyme and digestion time can significantly impact the results, making it a less reproducible method.
Some commonly used buffers and enzymes for enzyme-based antigen retrieval include:
- Trypsin: Trypsin is a commonly used enzyme for antigen retrieval in tissues such as muscle and skin. Trypsin is typically used in a buffer solution such as Tris-HCl or PBS.
- Pepsin: Pepsin is a commonly used enzyme for antigen retrieval in tissues such as stomach and esophagus. Pepsin is typically used in an acidic buffer solution such as 0.01M HCl.
- Collagenase: Collagenase is a commonly used enzyme for antigen retrieval in tissues containing collagen such as cartilage and bone. Collagenase is typically used in a buffer solution such as PBS.
- Proteinase K: Proteinase K is a commonly used enzyme for antigen retrieval in tissues such as brain and lung. Proteinase K is typically used in a buffer solution such as Tris-HCl.
pH-based retrieval: This method involves adjusting the pH of the buffer solution to make the antigens more accessible to antibodies.
Benefits: pH-based retrieval is a milder and less damaging method that can result in improved antigen detection.
Downsides: The choice of pH can significantly impact the results, making it a less reproducible method.
Some commonly used buffers and pH levels for pH-based antigen retrieval include:
- Tris-EDTA (TE) buffer at pH 9: This buffer is often used for retrieval of antigens in acidic tissues such as muscle or nerve.
- Sodium citrate buffer at pH 6: This buffer is often used for retrieval of antigens in formalin-fixed paraffin-embedded (FFPE) tissues.
- Tris buffer at pH 11: This buffer is often used for retrieval of antigens in basic tissues such as liver or spleen.
Antigen retrieval depends on the specific requirements of the IHC experiment and the type of tissue being analyzed. Each method has its own benefits and downsides, and it is important to carefully consider the best method for each specific case.