Tris Buffer Antigen Retrieval Protocol

Contents

  1. Introduction
  2. Materials
    1. Tris Buffer ( 10 mM TBS, 0.05% Tween 20, pH 10.0)
  3. Procedure
  4. References

Introduction

A Tris Buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues [1].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-link proteins, resulting a reduction in the available epitopes for antibody binding. Use of this basic antigen retrieval solution effectively unmasks some protein epitopes, but should be used with care as tissue may detach from slides somewhat more frequently than with other antigen retrieval solutions.

Materials

Tris Buffer ( 10 mM TBS, 0.05% Tween 20, pH 10.0)

  • Mix 1.21 g of Tris Base in 1000 ml of distilled water. Adjust the pH to 10.0 with 1 N NaOH and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract

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