In order to test tissue specimens with antibody, they first have to be preserved in fixative, embedded in paraffin, and sectioned very thinly onto glass microscope slides. Any piece of tissue, immediately after excision, must be placed into an adequate volume of fixative. Fixatives vary, but the standard one is 10% buffered formalin. After an optimum fixation time (for formalin, about 16 h), the sample must be embedded in paraffin and sectioned on a microtome. Paraffin-embedded sections placed on positively charged slides (either coated or commercially prepared) are then ready for various pretreatment steps. First, the paraffin must be replaced with water through a series of rehydration steps. Then, depending on the antigen to be tested, the section can be proteolytically digested with enzymes or heat-treated in low or high pH solutions. Following that, the endogenous peroxidase enzyme or oxidative compounds can be quenched in a hydrogen peroxide solution. The sections are then ready to be tested with antibody after an incubation in a normal serum solution blocks any available charged sites.
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