Figure 1. Indirect immunofluorescence staining of neurons in primary dissociated culture. Neurons from embryonic rat hippocampus were dissociated mechanically and by enzymatic treatment, plated in Petri dishes, and maintained up to 3 weeks in vitro using a conditioned medium.

(a) For staining ‘live’, cells were transferred into glucose-containing Ringer solution, processed for immunofluorescence staining using a guinea-pig antiserum against the GABA_A receptor ₂ subunit as primary antibody and goat anti-guinea-pig IgGs coupled to the red fluorescent carbocyanine dye, Cy3, and visualized by phase contrast and epifluorescence microscopy with a 63× water immersion lens. The primary antibody was directed against an extracellular epitope and therefore binds to GABA_A receptors inserted in the membrane. The image was captured using a charge coupled device (CCD) camera and depicts the superposition of the phase contrast and the fluorescence pictures captured separately. The red puncta on the soma and neurites of the immunopositive cell represent GABA_A receptors aggregated at postsynaptic sites.

(b) For double-immunofluorescence staining, cells were fixed with methanol (–20°C, 10 min) and incubated in a mixture of two primary antibodies (guinea-pig antiserum against the GABA_A receptor ₂ subunit and mouse monoclonal antibody against synaptophysin), followed by a mixture of two secondary antibodies (goat anti-guinea-pig IgGs conjugated to Cy3 and goat anti-mouse IgGs linked to Cy2). Images were captured by confocal laser scanning microscopy using a 100× oil immersion lens. The picture represents the superposition of the two fluorescence images captured simultaneously with distinct detectors. The green staining reveals presynaptic axon terminals immunopositive for synaptophysin and the red-staining postsynaptic GABA_A receptors mostly aggregated at sites of synaptic contact.