Mouse anti neun

On the 14th day post-surgery, rats were placed under deep anesthesia and infused with phosphate-buffered saline (PBS, 1 M, pH 7.4) and 4% paraformaldehyde. The lumbar spinal cord was harvested, frozen, and cut into 10-μm transverse sections, and the sections were placed on glass slides. Slides were washed with PBS three times, each for 10 min, and blocked with 5% donkey serum in PBS for 1 h. The slides were then incubated with primary antibodies: rabbit anti-IL-4Rα (1:200; Santa Cruz Biotechnology, Dallas, TX), mouse anti-NeuN (1:400; Cell Signaling Technology, Danvers, MA), goat anti-Iba-1 (1:400; Abcam, Cambridge, UK), and mouse anti-glial fibrillary acidic protein (GFAP) (1:400; Novus Biologicals, Littleton, CO) for 12 h at 4°C. The slides were washed with PBS and incubated with Dylight 488 donkey anti-rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA), Cy3 donkey anti-goat IgG (1:400; Abcam), or Dylight 549 donkey anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories) for 2 h at 4°C. The slides were washed, sealed with a coverslip using 4′,6-diamino-2-phenylindole Fluoromount-G (AmyJet Scientific, Wuhan, China), and visualized with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Images were acquired with a digital camera (Carl Zeiss) under the same light intensity for each stain.

At 6, 12, 24 or 48 h following HI, the cortices of the pups were harvested and cut into 5-mm sections for further analyses. Immunofluorescence was performed using the 24-h sections only. Sections were embedded with paraffin and incubated with rabbit anti-ASK1 polyclonal antibody (1:200; cat. no. ab131506; Abcam, Cambridge, UK) or rabbit anti-p-JNK monoclonal antibody (Thr183/Tyr185; 1:200; cat. no. 9255; Cell Signaling Technology, Inc., Beverly, MA, USA), and mouse anti-NeuN (1:150; cat. no. MAB377; EMD Millipore, Billerica, MA, USA) or mouse anti-GFAP (1:80; cat. no. MAB360; EMD Millipore) monoclonal antibodies. Subsequently, the sections were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit (1:120; cat. no. sc-2012; Santa Cruz Biotechnology, Inc.) or goat anti-mouse (1:120; cat. no. sc-2010; Santa Cruz Biotechnology, Inc.) immunoglobulin G (IgG), and tetramethylrhodamine-conjugated goat anti-rabbit (1:120; cat. no. sc-2780; Santa Cruz Biotechnology, Inc.) or goat anti-mouse (1:120; cat. no. sc-2781; Santa Cruz Biotechnology, Inc.) IgG. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, Inc., Dalian, China; 1:100). A total of 5 sections per rat were analyzed under a microscope (DTX500; Nikon Corporation, Tokyo, Japan).