Gold toning preserves integrity of silver enhanced immunogold particles during osmium tetroxide treatment for demonstration of a biogenic amine.
We describe a protocol that enhances immunolabelling of nervous tissue for ultrastructural study. Insect tissue is fixed, sectioned, and labelled with a polyclonal antiserum against serotonin and a secondary antibody conjugated with 1 nm colloidal gold. The gold particles are silver-enhanced to ease detection and then protected by gold toning. Finally, the tissue is post fixed in glutaraldehyde fixative followed by osmium tetroxide and further processed for electron microscopy. We demonstrated on insect nervous tissue that gold toning protects marker particles from the influence of osmium tetroxide. Use of buffered solutions throughout the protocol led to well preserved ultrastructural details, and marker particle size was not reduced with a short gold toning time. We also suggest use of this protocol for vertebrate or other invertebrate tissue.
Leitinger
G
G
Institut für Histologie und Embryologie, Karl-Franzens-Universität, Graz, Austria. gerd.leitinger@ncl.ac.uk
Pabst
M A
MA
Kral
K
K
D000818:Animals; D004396:Coloring Agents; D004210:Dissection / Q000379:methods; D017952:Ganglia, Invertebrate / Q000737:chemistry / Q000648:ultrastructure; D006046:Gold; D007150:Immunohistochemistry; D020050:Mantodea; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008867:Microtomy / Q000379:methods; D009420:Nervous System / Q000737:chemistry / Q000648:ultrastructure; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D009942:Organometallic Compounds; D009993:Osmium Tetroxide; D012701:Serotonin / Q000032:analysis*; D016622:Silver Staining / Q000379:methods*
D004396:Coloring Agents; D009942:Organometallic Compounds; C005460:uranyl acetate; D012701:Serotonin; D006046:Gold; D009993:Osmium Tetroxide