Optimization of Immuno-labeling and Counterstaining Abstract for Electron Microscopy

Image contrast of sections after immunolabeling should be well preserved to observe synaptic structures (i.e. cell membranes, synaptic vesicles which contain the neurotransmitter glutamate, post synaptic density (PSD), and synaptic cleft) in the brainstem clearly on the sections having immunogold particles with high labeling efficiency for the target protein. Immunolabeling Is used to label targeted proteins with gold particles (used due to their electron density and the fact TEM images are in black and white.) , EGFP was specifically targeted throughout these experiments. EGFP is expressed in the calyx of held terminals. The ultrastructure of synapses between the MNTB principal neuron (which localizes sound) and calyx of Held terminal can be preserved, defined, and imaged through the optimization of immunolabeling and counterstaining times to ensure high gold labeling efficiency and good contrast. Plastic embedded brain sections (70nm thickness) were incubated in blocking buffer, primary antibody, blocking, and secondary antibody, stabilized with 1% glutaraldehyde, and then stained with aqueous uranyl acetate (UA) and lead citrate (LC) for different times. Presynaptic terminals of the Calyx of Held were labeled with 12nm colloidal gold indicating the expression of the EGFP. By adding counterstaining with UA and LC, the lost contrast after immunolabeling was partially regained and the synaptic structures were clearly visualized. One minute staining of UA followed by one minute staining of LC gave the best contrast to the section.The most desirable images, which have the maximum amount of gold particles possible without sacrificing too much of the integrity of the synaptic contact’s ultrastructure, were obtained through optimization of the reaction times of both immunolabeling and counterstaining. It was found that in order to get high gold labeling efficiency the grid must be put through a three day immunolabeling reaction, and to retrieve the lost contrast counterstaining with 1min UA and 1min LC must be used.

Grids

Reagents

Grid box

Axon bundles

MNTB Principal neuron

N

Calyx of Held terminalCalyx of Held terminal (magnified)

Presynaptic Terminal

Postsynaptic Neuron