The primary antibodies used in this study have been used in previous reports. They included polyclonal guinea pig anti-GABA (1:1K, AB175; Chemicon, Temecula, CA),
29 polyclonal goat anti-CHAT (choline acetyltransferase, 1:100, AB144P; Chemicon),
36 rat anti-glycine antiserum (1:1000, a generous gift from David Pow, University of Queensland, Brisbane, QLD, Australia),
37 –39 polyclonal rabbit anti-GAD65 (glutamate decarboxylase, 1:1000, Chemicon),
40 monoclonal rat anti-GFAP (glial fibrillary acidic protein, 1:1000, Zymed Laboratory. Inc., South San Francisco, CA),
41 monoclonal mouse anti MHC II (major histocompatibility complex Class II molecules, 1:500; Chemicon),
42,43 monoclonal mouse anti-GS (glutamine synthetase, 1:1000; BD Transduction Laboratories, Palo Alto, CA),
39 and a mouse monoclonal antibody against Goα (1:1000, clone 2A; Chemicon).
44 The specificity of these primary antibodies has been demonstrated in previous studies and their staining patterns in our results were similar to the previous published data. Rabbit anti-5-HT antiserum was obtained from Immunostar Inc. (1:500, Hudson, WI). In the retinas that were not preincubated with 5-HT, 5-HT-immunoreactivity (IR) was absent in the neuronal somas in the GCL and the inner nuclei layer (INL), confirming the specificity of the 5-HT antibody. Cy3-conjugated and Cy-5 conjugated secondary antibodies were used at 1:200 dilution (Jackson ImmunoResearch, West Grove, PA). A fluorescent nuclear dye,
45 TO-PRO-3 (1:3000; Molecular Probe, Eugene, OR), was used to visualize the nuclei in the retinas, together with secondary antibodies.