TE antigen retrieval and immunostaining

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

Revision as of 14:49, 2 November 2009

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

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