Antigen retrieval: Difference between revisions

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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.
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.


== Protocol ==
== Types of retrieval methods ==
# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
=== Heat treatment ===
# Leave the chosen slides at room temperature just until the frost melts.
* Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding
# Hydrate slides in 1x PBS for 10 seconds.
* Specific protocol 1: citrate buffer [http://openwetware.org/wiki/Livesey:_Antigen_retrival][http://www.ihcworld.com/_protocols/epitope_retrieval/citrate_buffer.htm]
# Put slides into '''0.01 M Citric acid (pH 6.0), microwave''' just to a boil, and let cool for 5 minutes.
* Specific protocol 2: Tris-EDTA buffer [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm]
# 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


== Lab-specific protocols ==
=== Protease treatment ===
* Griffin Lab:
* Specific protocol 1: proteinase K [http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm]
* Specific protocol 2: trypsin [http://www.ihcworld.com/_protocols/epitope_retrieval/trypsin.htm]
 
=== Detergent treatment ===
* Specific protocol: SDS [http://openwetware.org/wiki/Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells]
 
== Personal and lab-specific protocols ==
* Korey Griffin's notes:
:* [[Griffin:Antigen Retrieval Technique]] - overview and comparison of techniques
:* [[Griffin:Antigen Retrieval Technique]] - overview and comparison of techniques
:* [[Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval]] - heat-based retrieval
:* [[Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval]] - heat-based retrieval
:* [[Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells|Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval]] - detergent-based retrieval
:* [[Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells|Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval]] - detergent-based retrieval
* [[Livesey: Antigen retrival]] - heat-based retrieval
* [[Livesey: Antigen retrival|Livesey's antigen retrival]] - heat-based retrieval
* [[Immunohistochemistry for AntiOsteocalcin Antibody]] - a protocol with a protease-based antigen retrieval
* [[Immunohistochemistry for AntiOsteocalcin Antibody]] - a protocol with a protease-based antigen retrieval


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* [[Antibodies]]
* [[Antibodies]]
* [[Griffin:Antibody Basics]]
* [[Griffin:Antibody Basics]]
== External links ==
* [[Image:3stars.png]] [http://www.ihcworld.com/_technical_tips/antigen_retrieval_tips.htm detailed description of antigen retrieval methods at IHCWorld]
* [[Image:3stars.png]] [http://www.ncbi.nlm.nih.gov/pubmed/17197287 Yamashita 2007] - 60 page detailed review article including antibody generation, fixation, and details on several antigen retrieval techniques
* [http://www.nordiqc.org/Techniques/Epitope_retrieval.htm review of retrieval methods at NordiQC and examples of which antibodies work with which method]
* [http://www.bioreagents.com/pages/protocols.cfm#3|Antigen retrieval protocols using heat at Thermo]
* [http://www.rockland-inc.com/commerce/misc/Antigen_retrieval_methods.jsp|Heat and protease antigen retrieval at Rockland, Inc.]
Commercial antigen retrieval kits:
* [http://www.bdbiosciences.ca/ptProduct.jsp?prodId=19647&catyId=76670 BD Pharmigen Retrievagen A pH 6] and [http://www.bdbiosciences.ca/ptProduct.jsp?prodId=19653&catyId=76670 BD Pharmigen Retrievagen B pH 9.5]
* [http://www.genetex.com/WebPage/Product/ProductDetail.aspx?CatalogItemID=33939 GeneTex antigen retrieval solution (heat-based)] and [http://www.genetex.com/WebPage/Product/ProductDetail.aspx?CatalogItemID=66695 GeneTex antigen retrieval solution (protease-based)]
* [http://www.abcam.com/10x-Heat-Mediated-Antigen-Retrieval-Solution-pH-6-0-ab973.html abcam heat-based antigen retrieval solution 10x]
* [http://www.biocompare.com/ProductListings/17837/Antigen-Retrieval-Solutions.html antigen retrieval search at biocompare]
[[Category:Protocol]]
[[Category:Antibody]]
[[Category:Protein]]
[[Category:In vitro]]

Latest revision as of 12:13, 2 November 2009

back to protocols

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

  • Specific protocol 1: proteinase K [4]
  • Specific protocol 2: trypsin [5]

Detergent treatment

  • Specific protocol: SDS [6]

Personal and lab-specific protocols

  • Korey Griffin's notes:

See also

External links

Commercial antigen retrieval kits:

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