Heat-induced antigen retrieval protocols

This protocol will generally produce good quality immunohistochemical staining. Frozen tissue sections normally do not need a heat-induced antigen retrieval step. 

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... 

Failure to achieve the optimal temperature required for heat induced antigen retrieval. When using a waterbath or steamer, allow sufficient time for the retrieval buffer to equilibrate to a temperature range of 95-99 °C. At high altitude (greater than-4,500 feet), the buffer will boil at less than 95 °C. Utilize a closed heating system such as a pressure cooker, autoclave or Pascal, or utilize a low temperature protocol if standardization of the validated procedure is not affected. 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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