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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
- Protease treatment
- Detergent treatment
Sample heat-treatment protocol using a citrate buffer
- Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
- Leave the chosen slides at room temperature just until the frost melts.
- Hydrate slides in 1x PBS for 10 seconds.
- Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
- Repeat Step 4 two more times (three times total).
- Rinse the slides 6 times in 1x TBST for 8 minutes each.
- Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
- Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
- Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
- Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
- Rinse 3 times in 1x TBS for 8 minutes each.
- Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
- Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
TBST = TBS + 0.1% Triton
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
- 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
- retrieval protocols using heat at Thermo
- and protease antigen retrieval at Rockland, Inc.
Commercial antigen retrieval kits: