Guinea pig anti viaat

Sections were cut at a thickness of 7 μm. After removing paraffin, sections were processed in a decloaking chamber (Biocare Medical, Concord, CA) using a citrate-buffer-based antigen retrieval medium (Biocare Medical) for 20 min at 110–115°C. They were then processed in PBS with 15% methanol and 0.3% H₂O₂ to block endogenous peroxidase activity. Aldehyde groups were removed by incubating the sections in sodium borohydride (1%) in PBS. After these treatments, the slices were incubated in a blocking PBS-based solution containing cold-water fish-skin gelatin (0.1%) and 0.1% Triton X-100. Tissues were then incubated overnight at 4°C with the following primary antibodies: chicken anti-GFP (1:1000 Aves labs, Tigard, OR), rabbit anti-GABAa receptor gamma 2 subunit (1:1500; Synaptic Systems, 224 003, Göttingen, Germany) mouse anti-GlyR (1:1000; Synaptic Systems, 146 011), guinea pig anti-VIAAT (1:1500; Synaptic Systems, data not shown). Primary antibodies were revealed by incubation for 2 h with secondary antibodies coupled to either Alexa Fluor-488 (Invitrogen, Saint Aubin, France) or DyLight 488, DyLight 549, and DyLight 649 (Jackson ImmunoResearch, Newmarket, UK). Sections were finally mounted using Prolong Gold Antifade Reagent (Invitrogen). For all experiments, control sections were incubated without primary antibodies.