Alkaline phosphatase APAAP method

Whereas antigen-retrieval technique serves to amplifying the immunocytochemical signal at the predetection phase, conventional methods of signal amplification, such as avidin biotin complex (ABC) and soluble enzyme-anti-enzyme immune complex techniques (peroxidase-anti-peroxidase complex and alkaline phosphatase-anti-alkaline phosphatase complex PAP and APAAP respectively), are applied in the phase of detection. For many years, the PAP and APAAP procedures represented the most sensitive and reliable and hence most popular techniques in many pathology laboratories. However, today these techniques are only rarely used, being substituted by modem more sensitive methods. 

Alternatively alkaline phosphatase-coupled secondary antibody can be used with an anti-alkaline phosphatase antibody (APAAP technique). For this method the following protocol may be used ... 

A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase-antiperoxidase), ABC (avidin-biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase-anti-alkaline phosophatase) techniques however,... 

The principles of the alkaline phosphatase-antialkaline phosphatase (APAAP) technique are the same as those described for the PAP method (Fig. 1.10), except that the PAP complex is replaced with an APAAP complex. The method has had three major applications (1) staining of tissues with high levels of endogenous peroxidase,... 

This reporter is obviously encountered in the APAAP technique, but it can also be conjugated to biotin and used successfully in the ABC methods. The enzyme is sourced from calf intestine, this form being resistant to the blocking effect of levamisole, which is added to the substrate buffer and blocks all other types of endogenous alkaline phosphatase activity. Several chromogens can be used, the most popular being Fast Red TR salt which yields a bright red alcohol soluble reaction product (34). 

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