There continue to be many new technical developments that propel advances in our understanding of biological events taking place at the cellular and molecular level. One method that developed early in the expansion of cell and molecular biology and still remains a valuable tool and major core laboratory protocol is immunoblotting. Immunoblot detection of proteins, originally and often called Western blotting, is a method that takes advantage of antibodies that are raised in animals following immunization with the target protein (immunogen). The ability of the generated antibodies to recognize the target protein with high specificity and bind with high affinity makes the method very powerful. When a cell- or tissue-derived mixture of proteins containing the target is first separated based on size or charge and then immobilized on an inert support, the method becomes very informative. This chapter presents aspects of the immunoblotting technique relevant to preparations of brain vascular proteins, standard blotting protocols for optimal detection and performance, and generation of chicken-raised polyclonal antibodies.