TE antigen retrieval and immunostaining

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(Tris EDTA heat-based antigen retrieval)
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=== Comments ===
=== Comments ===
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* Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.
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* 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 ==
== Immunostaining ==

Revision as of 14:31, 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.

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 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

  • 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
  • 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

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