Antigen retrieval

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(external links)
(External links)
Line 37: Line 37:
== External links ==
== External links ==
 +
* [[Image:3stars.png]] [http://www.ihcworld.com/_technical_tips/antigen_retrieval_tips.htm detailed description of antigen retrieval methods at IHCWorld]
* [http://www.bioreagents.com/pages/protocols.cfm#3|Antigen retrieval protocols using heat at Thermo]
* [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.]
* [http://www.rockland-inc.com/commerce/misc/Antigen_retrieval_methods.jsp|Heat and protease antigen retrieval at Rockland, Inc.]

Revision as of 11:02, 19 October 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.

Contents

Types of retrieval methods

  1. Heat treatment
  2. Protease treatment
  3. Detergent treatment

Sample heat-treatment protocol using a citrate buffer

  1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
  2. Leave the chosen slides at room temperature just until the frost melts.
  3. Hydrate slides in 1x PBS for 10 seconds.
  4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
  5. Repeat Step 4 two more times (three times total).
  6. Rinse the slides 6 times in 1x TBST for 8 minutes each.
  7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
  8. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
  9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
  10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
  11. Rinse 3 times in 1x TBS for 8 minutes each.
  12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
  13. 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:

See also

External links

Commercial antigen retrieval kits:

Personal tools