Cy3 labeled goat anti mouse

For immunofluorescence staining of spinal cord tissue, the spinal cord tissue was dehydrated in 30 % sucrose solution for 3 days, embedded in OCT, and cut into 10 μm thick sections in transverse section. Cryofixed spinal cord sections were permeabilized and blocked in TBST with 1 % BSA at ambient temperature for 1 h. The sections were then incubated overnight at 4 °C with the following antibodies: Anti-NeuN (1:300, Abcam, USA), Anti-CD68 (1:200, Abcam, USA), Anti-iNOS (1:200, Abcam, USA), and Anti-Arg1 (1:200, Abcam, USA). To visualize the sections, Cy3-labeled goat anti-mouse (1:500, Beyotime, China) and Alexa Fluor 488 goat anti-rabbit (1:500, Beyotime, China) were used as secondary antibodies. Nuclear detection was performed by applying Fluoroshield Medium containing DAPI (Abcam, USA). Finally, confocal microscopy was used to capture images of the sections.

The DDIT4 (rabbit) antibody (#10,638–1-AP, 1:1000 dilution) and β-actin (mouse) antibody (#66,009–1-Ig, 1:5000 dilution) were obtained from Proteintech (Chicago, IL, USA). Anti-NF-κB p65 (#6956, 1:1000 dilution) and anti-phospho-p65 (#3033, 1:1000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA). Cy3-labeled goat anti-mouse (#A0521, 1:200 dilution) and FITC-labeled goat anti-rabbit antibodies (#A0562, 1:200 dilution) and DAPI were obtained from Beyotime Institute of Biotechnology (China). The DDIT4-AS1 antisense oligonucleotides (ASO) and the control ASO were purchased from Integrated Biotech Solutions Co., Ltd. (Shanghai, China); the sequences are listed in Table S2. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 plasmid pYSY-spCas9-sgRNA-Puro was obtained from YSY Biotech (Nanjing, China). The transfection reagent jetPRIME was purchased from Polyplus Transfection (Illkirch, France). The RNA polymerase II transcription inhibitor α-amanitin was purchased from Medchem Express (Princeton, NJ, USA). RNAse A + T cocktail was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The super electrochemiluminescence (ECL) kit was obtained from US Everbright Inc. (Suzhou, China).