Staining methods heat-induced antigen retrieval

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. 

Demasking protocol is inappropriate or has been omitted. Some tissue antigens require proteolytic enzyme digestion or heat induced antigen retrieval performed prior to staining. The need for pretreatment depends on the type and extent of fixation, specific characteristics of the antigen and the type of antibody used. Use the pretreatment method recommended by the manufacturer. No single pretreatment is suitable for all applications. 29-33, 51-65... 

The staining protocol and differences in pretreatment methods are an important factor when formalin-fixed and paraffin-embedded material is nsed, as not all antibodies work satisfactorily under these conditions. Several different approaches can be found in the literature. Heat-induced antigen retrieval by the use of microwave heat treatment has been demonstrated to be more effective in labeling intraneuronal Ap compared to no or enzymatic pretreatment [29]. Information on the use of FA is inconsistent. It is widely used to increase the immunoreactiv-ity of amyloid plaques in AD however, it has been shown that the effect on intraneuronal Ap is low and that use of FA sometimes results in a loss of cellular detail [30]. A recent study tried to quantify the effect of FA pretreatment and analyzed plaque load and intraneuronal Ap immunoreactivity using an APi, antibody in 6-month-old APP/PSIKI mice (Fig. 3 reproduced from [25] with permission from Elsevier). 

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