TE antigen retrieval and immunostaining
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== Immunostaining ==
== Immunostaining ==
Revision as of 14:34, 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.
- 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
- 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
Tris-EDTA Buffer final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:
(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.
- 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
- 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.
Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.