Improved immunocytochemical staining through the use of Fab fragments of primary antibody, Fab-specific second antibody, and Fab-horseradish peroxidase.

A modification of the unlabeled antibody method of immunocytochemistry is described here that offers increased immunoreagent penetration and greatly reduced background staining. The method involves the following alterations to the conventional technique; the use of Fab fragments of primary antibody, rather than whole immunoglobulin G (IgG) or serum; the use of a second, or link, anti-rabbit IgG serum that is Fab fragment-specific, rather than directed against the whole rabbit IgG molecule; the use of the Fab--horseradish peroxidase complex described by JR Slemmon, PM Salvaterra, and K Saito (J Histochem Cytochem 28:10, 1980), rather than peroxidase--antiperoxidase (PAP). Steps 2 and 3 alone brought about a significant reduction in background staining, but did not increase the depth of immunostaining, as compared to the PAP technique. When all three steps were combined, however, background staining was further reduced, and there was a five- to tenfold increase in the depth of immunostaining. These readily made changes should be useful in preembedding immunocytochemistry whenever enhanced reagent penetration is required.