**6. Effects of diluents on antibodies in immunohistochemistry**

The relationship between epitopes and paratopes of antibodies is thought to be similar to that between keys and keyholes. However, since these structures change their conformations to form a final specific and tight binding after antigen-antibody association, conservation of the flexibility of their polypeptide chains should be important. Although hydrogen bonds, hydrophobic forces, electrostatic forces, and van der Waals forces all participate in the final tight binding, electrostatic forces are important for the initial contact and association of antigen and antibody molecules (i.e., the net charges of each molecule and the neighboring charges of antigens). Buffer type, ionic strength, pH, and the presence of detergents in solutions are likely to exert strong influences on the antigen-antibody reaction. Although many kinds of diluents are commercially available for immunohistochemistry and Western blotting and yield good results with low background staining and a high sensitivity for some antigens, systematic studies of antibody diluents for immunohistochemistry have not been performed. In this section, the effects of dilution solutions for primary antibodies on immunostaining for light and electron microscopy are described.
