What is Enzymatic Retrieval?
Enzymatic retrieval is a technique used in
histology to unmask antigens in tissue sections, making them more accessible for antibody binding during immunohistochemistry (IHC) procedures. This process is essential for improving the sensitivity and specificity of antibody-antigen interactions.
Why is Enzymatic Retrieval Important?
Tissue fixation, commonly done using
formalin, cross-links proteins and other molecules, which can obscure antigenic sites. Enzymatic retrieval helps to reverse these cross-links, thus increasing the effectiveness of the staining process. This is particularly crucial for detecting specific
biomarkers in diagnostic pathology.
How Does Enzymatic Retrieval Work?
The method involves treating tissue sections with specific enzymes that can digest proteins and break down the cross-links formed during fixation. Commonly used enzymes include
proteinase K,
trypsin, and
pepsin. The choice of enzyme and its concentration, as well as incubation time, depends on the type of tissue and the antigen of interest.
Deparaffinization: Remove paraffin from tissue sections to make them more permeable.
Rehydration: Rehydrate tissue sections through a series of graded alcohol solutions.
Incubation with Enzyme: Treat the sections with the chosen enzyme solution at a specific temperature for a defined period.
Rinse: After incubation, rinse the sections to remove the enzyme.
Proceed with IHC: Apply primary and secondary antibodies, followed by visualization techniques.
Improved antigen accessibility, leading to stronger and more specific staining.
Compatibility with a wide range of tissues and antigens.
Flexibility in optimizing conditions based on tissue and antigen type.
Over-digestion can lead to tissue damage and loss of morphology.
Not all antigens respond well to enzymatic retrieval; some may require alternative methods like
heat-induced epitope retrieval (HIER).
Optimization can be time-consuming and might require multiple trials.
Enzyme Selection: Choose the appropriate enzyme for the tissue and antigen.
Concentration: Determine the optimal concentration of the enzyme.
Incubation Time and Temperature: Optimize incubation time and temperature to achieve best results without damaging tissue morphology.
Control Samples: Use control samples to validate the effectiveness of the retrieval process.
What are Alternative Methods?
Besides enzymatic retrieval, other methods include
HIER and
chemical retrieval. HIER uses heat to break the cross-links, while chemical retrieval employs strong acids or bases. Each method has its own set of advantages and limitations, and the choice depends on the specific requirements of the study.
Conclusion
Enzymatic retrieval is a vital technique in histology for enhancing antigen detection in tissue sections. Understanding its principles, advantages, and limitations allows for effective optimization and application in various histological studies. As with any technique, careful consideration and optimization are key to achieving reliable and reproducible results.
