TE antigen retrieval and immunostaining

From OpenWetWare

Revision as of 13:49, 2 November 2009 by Jakob Suckale (Talk | contribs)
Jump to: navigation, search

This 2-part protocol describes antigen retrieval with Tris EDTA buffer followed by immunostaining. Antigen retrieval can be helpful for aldehyde-fixed, paraffin-embedded samples that don't give sufficient signal with a standard immunostaining protocol. Several antigen retrieval methods exist but, unfortunately, you need to determine empirically which one is most suited to your antibody.

Contents

Deparaffinisation

Tris EDTA heat-based antigen retrieval

Principle

  • aldehyde treatment (paraformaldehyde, glutaraldehyde) chemically fixes cells and tissue
  • paraffin-embedding allows very thin tissue sections to be cut
  • however, some antibodies don't work well after aldehyde fixation and paraffin embedding as opposed to other fixatives and cryosections
  • antigen retrieval methods can improve antibody binding by reversing chemical modification of epitopes or by unfolding/refolding epitopes

Material

Steps

Comments

Immunostaining

Personal tools