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.

Deparaffinisation

• deparaffinise sections in xylene for 10min
• hydration series

• 2 changes of 100% ethanol for 3 minutes each
• 95% ethanol 1min
• 80% ethanol 1min
• distilled water

Note: Incubation times and repeats vary between protocols.

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 with adjustment. Store the solution at room temperature for several weeks or at 4ºC for months.

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

Steps

• pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil)
• immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody)
• remove the container and allow to cool at RT for 20 minutes
• rinse sections in PBS Tween 20 for 2x2 min

Comments

• Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.
• Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer.

Immunostaining

Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.

See also

• Tris EDTA antigen retrieval at IHCWorld
• Antigen retrieval page at abcam
• Antigen retrieval page at Millipore
• Google Books search for "tris edta antigen retrieval"