Immunocytochemical Techniques

One of the most powerful tools in the identification of various neuronal types and then connectivities is immunocytochemical localization of specific neuronal markers, e g., synthetic enzymes for neurotransmitters at precise cellular and subcellular levels. Several methods exist for the localization of tissue antigens, at both the light and electron microscopic (EM) levels. The fluorescein-labeled antibody or immunofluorescent method developed by (1958 ) for the localization of tissue antigens with the light microscope has been employed for the localization of enzymes involved in the metabolism of neurotransmitters such as dopamine-β-hydroxylase (Hartman, et al., 1972 ; Hartman, 1973 ), tyrosine hydroxylase (Pickel et al., 1975a ), phenylethanolamine-N -methyltransferase (Hokfelt et al., 1974 ), DOPA decarboxylase (Hokfelt et al, 1973 ), and L-glutamate decarboxylase (GAD) (Kataoka et al., 1984). There are some deficiencies in this otherwise sensitive and specific method, however, such as the masking of the specific fluorescence by the inherent background fluorescence of tissue, and a lack of permanence of the preparations. (1966 ), (1966 ) reported that enzymes of small molecular weight, such as acid phosphatase or peroxidase, could be conjugated to antibodies by bifunctional reagents.