Localization of GABA and glycine in goldfish retina by electron microscopic postembedding immunocytochemistry: improved visualization of synaptic structures with LR white resin.
A post-embedding, electron microscopic immunocytochemistry technique, modified from existing protocols, was used to examine the labelling patterns of GABA immunoreactivity and glycine immunoreactivity in goldfish retina. Retinae were fixed in mixed aldehyde solution, dehydrated in ethanol, stained en bloc with uranyl acetate and phosphotungstic acid and embedded in LR White resin. Substances were localized in thin sections by floating grids first on a drop of primary antiserum and then on a colloidal gold-IgG conjugate. Finally, grids were exposed to osmium vapour. The localization of GABA immunoreactivity matched that of [3H]-GABA uptake or glutamate decarboxylase immunoreactivity as described previously. In the outer retina, GABA immunoreactivity was found in the cell bodies and axon terminals of H1 horizontal cells and their dendrites opposite cone photoreceptor terminals. Selected amacrine cell bodies were labelled, as were many processes, both synaptic and non-synaptic, throughout the inner plexiform layer, including most amacrine cell processes contacting the synaptic terminals of type Mb bipolar cells. Numerous amacrine cells, their processes in the inner and outer plexiform layers, and photoreceptor terminals contained glycine immunoreactivity in a distribution similar to that shown by [3H]-glycine uptake. Despite the absence of osmium in the primary or secondary fixative, our protocol results in excellent visibility of synaptic structures and detectability of the colloidal gold immunolabel. Also, it does not cause extraction of the HRP/DAB reaction product and is therefore suitable for double-label analysis of neurons labelled with horseradish peroxidase.
Studholme
K M
KM
Department of Neurobiology and Behavior, State University of New York, Stony Brook 11794.
Yazulla
S
S
D000180:Acrylic Resins; D000818:Animals; D003530:Cyprinidae / Q000378:metabolism*; D005998:Glycine / Q000032:analysis*; D006054:Goldfish / Q000378:metabolism*; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D012160:Retina / Q000032:analysis*; D013569:Synapses / Q000648:ultrastructure*; D005680:gamma-Aminobutyric Acid / Q000032:analysis*
D000180:Acrylic Resins; D005680:gamma-Aminobutyric Acid; C048707:LR white; D005998:Glycine