BACKGROUND: In this report, we describe a new quantitative electron microscopicprotocol based on the use of double colloidal-gold post-embedding immunostaining procedure as markersto analyze the subcellular distribution of enkephalin (ENK) and GABA neurotransmitters simultaneouslyin the same ultrathin tissue sections in the periaqueductal gray area (PAG) of the rat brain.
MATERIALAND METHODS: Double gold particle signals were significantly improved using a number of technical adjustmentsfor the immunogold electron microscopic co-localization technique of ENK and GABA. The GABA-like neuronalelements were immuno-reacted with 20 nm gold particles and the enkephalin-like immunoreactive neuronswere labeled with 10 nm gold particles. This double labeling was more apparent in tissue sections thatwere deactivated for the gold staining of the first antibody. Excellent double labeling was obtainedwhen we blocked antigenicity of the first antiserum with hot (80 degrees C) paraformaldehyde fumes. Tominimize the clumping of the second gold particles around the first gold particles used against the firstantibody, we tried different staining order for the neurotransmitters tested in this study. It was necessaryto use a detergent (Triton X-100) at very low concentration (0.1%) instead of etching to expose the antigenicdeterminants of the neurotransmitters and at the same time to reduce the deleterious effects on the morphologyof the tissue sections. Furthermore, the high-glutaraldehyde fixation and the decrease in the intervalbetween cutting and labeling of the ultrathin sections significantly improved the results obtained inthis study.
RESULTS: Double labeling of sections with ENK and GABA produced co-localization in 23.1%and 1.2% of the immunoreactive axonal terminals and dendrites, respectively. Most of the double-labeledterminals contained more GABA-like than ENK-like immunolabeling. Half of the axon terminals [51%] anddendrites [56%] in the ventrolateral PAG were not labeled with either of GABA or ENK immunoreactivity.
CONCLUSIONS: This procedure was found to be completely compatible with good double immunolabeling andultrastructural preservation.

Keywords: central gray, Pain, GABA, electron microscopy, gold particles