Antigen retrieval
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Detection techniques using antibodies often fail to work on PFA- or formalin-fixed, paraffin-embedded sections. Antigen retrieval methods can then, in some cases, enable specific antibody detection. They work by reversing some of the chemical modification of epitopes during fixation. These procedures will not help much with epitope loss due to denaturation during sample treatment, like hot paraffin embedding.
Types of retrieval methods
Heat treatment
- Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding
- Specific protocol 1: citrate buffer [1][2]
- Specific protocol 2: Tris-EDTA buffer [3]
Protease treatment
Detergent treatment
- Specific protocol: SDS [6]
Personal and lab-specific protocols
- Korey Griffin's notes:
- Griffin:Antigen Retrieval Technique - overview and comparison of techniques
- Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval - heat-based retrieval
- Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval - detergent-based retrieval
- Livesey's antigen retrival - heat-based retrieval
- Immunohistochemistry for AntiOsteocalcin Antibody - a protocol with a protease-based antigen retrieval
See also
External links
detailed description of antigen retrieval methods at IHCWorld- Yamashita 2007 - 60 page detailed review article including antibody generation, fixation, and details on several antigen retrieval techniques
- review of retrieval methods at NordiQC and examples of which antibodies work with which method
- retrieval protocols using heat at Thermo
- and protease antigen retrieval at Rockland, Inc.
Commercial antigen retrieval kits:
