Factors Affecting Antigen Retrieval

Many factors influence antigen retrieval, including fixation, heating, retrieval fluid, and antibodies. 

Tissue specimens ideally should be placed in the fixative immediately after their removal from the body. This problem arises in studies of human tissues, for their immediate fixation is usually not feasible. If immediate fixation is not possible, the tissue must be kept cool and moist by covering it with a piece of cloth soaked in sterile, cold saline for not more than 20-30 min. During this time the specimen should not contact any dry and absorbent object such as paper, a paper towel, or gauze. To keep the paper trail of the specimen (source, time, place of collection, etc.) is no less important. 

Note that even human tissues fixed immediately after their removal from the body may undergo cellular changes because usually the vascular supply is terminated before the tissue is surgically removed. During this duration ( 1 hr) the tissue remains at body temperature, at which the activity of digestive enzymes continues, damaging the cellular structures (Grizzle et al., 2001). 

Chemical fixation is not the only factor that causes loss or irreversible masking of antigens. Treatments such as dehydration and embedding following fixation also play a role in the loss of immunorecognition of antigens. Absolute ethanol and xylene must not contain traces of water, and the water bath should be very free from contaminants such as bacteria, fungi, dust, and dirt. Once the sections are contaminated, they cannot be decontaminated. 

On the basis of antigen retrieval obtained with protein denaturing agents, it has been proposed that in certain cases antibodies recognize denatured but not native antigens. 

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