Antigen retrieval incubation

For antigen retrieval, incubate tissue slides in a preheated 10 mM citrate solution in a 95 °C water bath for 20 min. 

In 1991 Shi et al. published their seminal observation that high-temperature incubation of formalin-fixed, paraffin-embedded (FFPE) tissue sections in buffers for short periods led to improved immunohistochemical staining.1 However, more than 15 years later, heat-induced antigen retrieval (AR) remains largely an empirical procedure, requiring the optimization of several critical parameters by trial and error.2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method. 

Boenisch T. Heat-induced antigen retrieval restores electrostatic forces prolonging the antibody incubation as an alternative. Appl. Immunohistochem. Mol. Morphol. 2002 10 363-367. 

It is possible to restore antigenicity in tissue sections by digesting the rehydrated sections with any of several proteolytic enzymes, or by incubating the tissue section at elevated temperatures in any of several liquids, including water and various buffers (antigen retrieval).12... 

The effectiveness of antigen retrieval depends on the specifics of fixation, dehydration and embedding, the nature of the protein to be detected, and the pH, ionic strength, and temperature of the incubation buffer, as well as the length of incubation. 

Antigen retrieval using a domestic vegetable steamer or a water bath Preheat steamer or water bath with a Coplin jar filled with an antigen retrieval solution of choice until temperature reaches 95°C 100°C. Immerse slides in the Coplin jar and place the lid loosely on the jar. Incubate for 30 60 min and turn off the steamer or water bath. Place the Coplin jar at room temperature and allow the slides to cool for 20 min. 

Overdigestion of tissue is common when proteinase-K or triton is used to improve antigen retrieval penetrance of the primary antibody. The easiest correction is to dilute triton solutions and decrease the time of the proteinase-K incubation. Tissue can also be digested when the hydrogen peroxide solution, used to quench endogenous peroxidase activity, is left on too long or is too concentrated. To correct this, check... 

In contrast to the EPOS system, a modification of the highly sensitive two-step immunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kammerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. 

This new definition of immunocytochemistry derives from advances in antibodylabeling methods in recent years. These advances resulted from specific needs in animal research. Initially, formalin-fixed paraffin sections were used for immuno-histochemistry however, results were inconsistent. In most cases, the antibody did not label anything or it labeled too many cells and was dubbed over fixed. This problem led to the development of the epitope retrieval or antigen retrieval methods, where sections of tissue are treated with heat in buffers before antibody incubations. Unfortunately, epitope retrieval methods can be unique from antibody to antibody and also, for the same antibody, from tissue to tissue. Epitope retrieval is complicated and best avoided. For animal research, a simple method was then developed where tissue was fixed in paraformaldehyde and not formalin or alcohol and subsequently frozen sections were cut on a cryostat. This eliminated the steps of dehydration, embedding in paraffin, rehydration after sectioning, and epitope retrieval before antibody incubation. This was a major breakthrough. 

Tissue specimens are fixed in formalin and subsequently embedded in paraffin in the majority of cases. While formalin fixation preserves tissue morphology, it also alters the three-dimensional structure of tissue proteins. This alteration can result in a modification of the antigen s epitopes and/or of its electrostatic charges (EC). The loss of an epitope results in an antigen s inability to react with the paratope of the antibody and can only be corrected by the restoration (retrieval) of the epitope. The reduction of net negative ECs of antigens decreases the avidity of the immune reaction and may be compensated for by prolonging the incubation time with the primary antibody (1) or the use of different antibody diluents (2). 

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