TE antigen retrieval and immunostaining

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.

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)

• 0.5ml Tween 20 final 0.05% (for 1x)

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.