Silver-enhanced diaminobenzidine-sulfide (SEDS): a technique for high-resolution immunoelectron microscopy demonstrated with monoamine immunoreactivity in monkey cerebral cortex and caudate.
A common frustration of immunoelectron microscopy (IEM) is the density of the 3,3'-diaminobenzidine (DAB) label, which obscures intracellular details of labeled structures. To overcome this problem, a silver enhancement protocol was developed which leaves silver deposits on very low levels of DAB. The resulting label is composed of easily visualized punctate silver deposits, localized in processes with little or no detectable DAB. This technique incorporates several modifications into previously described methods for silver enhancement of DAB. The principal innovation is to pretreat the DAB label with sodium sulfide before silver enhancement, which substantially increases the sensitivity of the silver enhancement. In addition, cysteine was used in place of thioglycolic acid to suppress tissue argyrophilia, allowing use of both glutaraldehyde- and paraformaldehyde-fixed tissue without degradation of ultrastructure. We demonstrate this technique with dopamine, norepinephrine (NE), and serotonin (5HT) immunoreactivity in monkey prefrontal cerebral cortex and with dopamine immunoreactivity in the anterior caudate. The punctate label allows essentially unobscured visualization of the intracellular details and cell membranes of these monoamine axons. Whereas 5HT axons formed small asymmetric synapses, dopamine and NE axons typically formed small symmetric synapses with notably subtle membrane specializations. It is likely that these are often obscured by conventional DAB labeling. The use of several preparations indicates that this technique will be useful with a variety of antibodies. It might also provide an attractive alternative to colloidal gold, especially with glutaraldehyde-fixed tissue which is not easily penetrated by gold particles.
Smiley
J F
JF
Section of Neurobiology, Yale School of Medicine, New Haven, CT 06510.
Goldman-Rakic
P S
PS
D015100:3,3'-Diaminobenzidine; D000818:Animals; D001369:Axons / Q000737:chemistry / Q000648:ultrastructure; D015306:Biogenic Monoamines / Q000032:analysis*; D002421:Caudate Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002540:Cerebral Cortex / Q000737:chemistry* / Q000648:ultrastructure; D004298:Dopamine / Q000032:analysis; D006801:Humans; D008253:Macaca mulatta; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009638:Norepinephrine / Q000032:analysis; D012701:Serotonin / Q000032:analysis; D016622:Silver Staining / Q000379:methods*; D013569:Synapses / Q000737:chemistry / Q000648:ultrastructure; D013997:Time Factors
D015306:Biogenic Monoamines; D015100:3,3'-Diaminobenzidine; D012701:Serotonin; D004298:Dopamine; D009638:Norepinephrine