Ab109223

Protein expressions of GAP-43 (8945, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), ST2 (ab25877, 1:2,000; Abcam), IL-33 (ab207737, 1:2,000; Abcam), CD16 (ab109223, 1:2,000; Abcam), and CD206 (sc-58986, 1:1,000; Santa Cruz Biotechnology) were determined in spinal cord homogenates by western blot analysis as previously described [25] . After the protein concentration was measured using bovine serum albumin, the proteins were separated by SDS-PAGE and then transferred onto a polyvinylidene di uoride membrane. Next, the membranes were blocked with 5% nonfat milk for 60 min and incubated with primary antibodies for 12 h at 4° C. Finally, the membranes were incubated with secondary antibody (A32723, A32754, 1:5,000; Invitrogen) for 1 h, and images were acquired (BioSpectrum 515; LabMode, Borehamwood, UK). Image J (National Institutes of Health, Bethesda, MD, USA) was used to visualize the reaction products for quantifying protein expression.

Frozen sections (20 mm) were permeabilized three times with phosphate-buffered saline/Tween 20 (PBST) for 15 min, blocked with 10% goat serum for 30 min, and incubated overnight with primary antibody to NeuN (ab104224, 1:200: Abcam, Cambridge, UK), cleaved caspase-3 (ab49822, 1:200; Abcam), Iba I (01919741, 01226723, 1:100; Wako, Osaka, Japan), ST2 (ab25877, 1:100; Abcam), CD16 (ab109223, 1:100; Abcam), or CD206 (sc-58986, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the sections were washed in PBST for 30 min and incubated with secondary antibody (A32723, A32754, 1:100; Invitrogen, Carlsbad, CA, USA) for 8 h. Finally, sections were washed in PBST for 30 min and then incubated with 4′,6-diamidino-2-phenylindole for 30 s. The stained sections were analyzed in elds/sections with a confocal laser scanning microscope (Nikon).