Anti asic1

The immunofluorescence procedures were according to the article published previously.³² The thickness of the spinal cord slice was 14 μm. The primary antibodies incubated on the spinal cord included anti‐ASIC1 (1:50, Alomone Labs, Jerusalem, Israel), anti‐NeuN (1:50, Merck Millipore, Darmstadt, Germany), anti‐GFAP (1:100, Cell Signaling Technology, Danvers, MA, USA), and anti‐CD11b (1:50, Bio‐Rad, California, USA). The secondary antibodies were Alexa Fluor 488 (1:500, Molecular Probes New York) and Alexa Fluor 555 (1:100, Molecular Probes New York). Fluorescence in situ hybridization (FISH) was performed with enhanced sensitive ISH Detection Kit I (POD, Boster MK1030, Wuhan, China). A locked nucleic acid probe with complementarities to miR‐485 was labeled with 5′‐ and 3′‐digoxigenin and synthesized by Exiqon. The detailed method was described in an article published in our laboratory.²⁵

Rats were injected with tribromoethanol (200 mg/kg, i.p.) for anesthesia, and transcardially perfused with isotonic 0.1 M phosphate buffer (pH 7.4), followed by isotonic 4% PFA. Then, the brain was fixed in 4% PFA at 4°C overnight and cryoprotected in 20–30% sucrose in 0.1 M phosphate buffer. Briefly, sections (20 μm) were fixed with 4% PFA and washed with 0.3% Triton X-100/PBS. Subsequently, the sections were incubated for 1 h in blocking serum (5% normal donkey serum in 0.2% Triton-X 100/PBS) at room temperature and then with primary antibodies (anti-ASIC1, Alomone Labs, Jerusalem, Israel; anti-Parvalbumin, Swant, Marly1, Switzerland) at 4°C overnight. Finally, the sections were washed with PBS and incubated with species-matched secondary antibodies (Alexa 488-conjugated anti-mouse IgG; Alexa 546-conjugated anti-rabbit IgG, Invitrogen) at room temperature for 1 h. The images were captured using the Evos FL auto 2 microscope (Invitrogen).

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.