1. Optimization of Immuno-labeling and Counterstaining Abstract for Electron Microscopy
Image contrast of sections afterimmunolabelingshouldbe well preserved toobserve synapticstructures(i.e.
cell membranes, synapticvesicles whichcontainthe neurotransmitterglutamate,postsynapticdensity(PSD),and
synapticcleft) inthe brainstem clearly onthe sectionshavingimmunogoldparticles withhighlabelingefficiency forthe
target protein. ImmunolabelingIsusedtolabel targetedproteinswithgoldparticles(useddue totheirelectrondensity
and the fact TEM imagesare in blackand white.) ,EGFPwas specificallytargetedthroughoutthese experiments.EGFPis
expressedinthe calyx of heldterminals. The ultrastructure of synapses betweenthe MNTBprincipal neuron (which
localizessound) andcalyx of Heldterminalcanbe preserved,defined,andimagedthroughthe optimizationof
immunolabelingandcounterstaining timestoensure highgoldlabelingefficiencyandgoodcontrast.Plasticembedded
brainsections(70nmthickness) were incubatedinblockingbuffer,primaryantibody,blocking, andsecondary antibody,
stabilizedwith 1%glutaraldehyde,andthenstainedwith aqueousuranyl acetate (UA) andleadcitrate (LC) fordifferent
times.Presynapticterminalsof the Calyx of Heldwere labeledwith12nmcolloidal goldindicatingthe expressionof the
EGFP. By addingcounterstainingwithUA andLC, the lostcontrast afterimmunolabelingwas partially regainedandthe
synapticstructureswere clearlyvisualized. One minutestainingof UA followedbyone minute stainingof LCgave the
bestcontrast to the section.The mostdesirableimages,whichhave the maximumamountof goldparticlespossible
withoutsacrificingtoomuchof the integrityof the synapticcontact’sultrastructure,were obtainedthrough
optimizationof the reactiontimesof bothimmunolabelingandcounterstaining.Itwasfoundthatinorderto get high
goldlabelingefficiencythe gridmustbe put througha three dayimmunolabelingreaction,andtoretrieve the lost
contrast counterstainingwith1minUA and 1minLC mustbe used.
Grids
Reagents
Grid box
Axon bundles
MNTB Principal neuron
N
Calyx of Held terminal
Calyx of Held terminal (magnified)
Presynaptic Terminal
Postsynaptic Neuron