Immunofluorescent labeling was performed as previously described (Sun et al., 2019 (link)). Rats were transcardially perfused with 0.9% normal saline and 4% paraformaldehyde, and the spinal cord was removed and post-fixed for 2 h. The thickness of the spinal cord slice 20 μm was processed. After blocking in phosphate-buffered saline (PBS) containing 7% normal donkey serum, 0.3% Triton X-100, and 0.05% sodium azide at room temperature for 1 h, the slices were incubated with primary antibodies, including anti-ASIC1 (1:50, Alomone Labs, Jerusalem, Israel), anti-NKCC1 (5 μg/ml, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-NeuN (1:50, Merck Millipore, Darmstadt, Germany), anti-GFAP (1:100, Cell Signaling Technology, Danvers, MA, USA) or anti-CD11b (1:50, Bio-Rad, CA, USA) overnight at 4°C. After wash, the slices were then incubated in secondary antibody included Alexa Fluor 488 (1:500, Molecular Probes New York) or Alexa Fluor 555 (1:100, Molecular Probes New York) for 2 h at room temperature.
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