The Choice of Secondary Antibodies

Secondary antibodies are offered in three different forms whole IgG, F(ab )2 fragments, and Fab fragments. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures, and is the most cost-effective. 

F(ab )2 fragments of antibodies are generated by pepsin digestion of whole IgG antibodies to remove a portion of the Fc region (see Sect. 1.1). Elimination of nonspecific binding of the Fc region of antibodies to Fc receptors on cell surfaces in 

---

The choice of secondary antibody is between a fluorescently labeled antibody or an antibody that leaves a histochemical deposition for the detection of the BrdU, such as alkaline phosphatase (AP) deposition of nitroblue tetrazolium (NBT) or horseradish peroxidase (HRP) deposition of diaminobenzidine (DAB). These histochemical detection methods are about ten times more sensitive than that of normal fluorescently labeled antibodies. However, one consequence of the chemical deposit is to quench any costaining with a fluorescently labeled antibody in the region of BrdU label. Both the AP reaction, which leaves a blue precipitate, and the HRP reaction, which leaves a brown precipitate, for a double-label could be used. One shortcoming of this approach is the difficulty in detecting subcellular localization, or the colocalization of two targets. Both of these methods are outlined in this protocol. 

---