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After being blocked in the buffer (containing 0.5% Triton X-100, 5% donkey serum in PBS) for 1 hr at room temperature, they were incubated in primary antibodies, including anti-glutamate (1:500, rabbit, Sigma), anti-GABA (1:500, rabbit, Sigma), anti-ChAT (1:500, goat, Millipore),anti-mu opioid receptor (1:500, rabbit, Abcam), and anti-TUBB3 (1:500, rabbit, Abcam) at 4 C for 24 hr