TE antigen retrieval and immunostaining

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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

Tris-EDTA Buffer final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:

  • 1.21g Tris final 10mM
  • 0.37g EDTA final 1mM
  • distilled water to 1000 ml

(or 100 ml to make 10x)

The pH is typically around 9.0. Store the solution at room temperature for 3 months or at 4ºC for longer.

This buffer is basically a TE buffer with added Tween 20.

Steps

Comments

Immunostaining

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