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.

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: (or 100 ml to make 10x)
 * 1.21g Tris final 10mM
 * 0.37g EDTA final 1mM
 * distilled water to 1000 ml
 * 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.