Ab49999

Distal colon tissue was homogenized in RIPA buffer (P0013B; Beyotime, Hangzhou, China) with phosphatase inhibitors (S1873; Beyotime), and protein concentration was determined using the bicinchoninic acid assay method. 20 micrograms of total protein that was extracted from 100 mg colonic mucosa was separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). After the membranes were blocked with 5% skimmed milk in Tris buffer saline–Tween 20 (TBST), membranes were immunoblotted with the primary antibody against vascular endothelial growth factor (VEGF) (ab46154; Abcam, Shanghai, China), hypoxia-inducible factor (HIF)-1α (ab2185; Abcam), inducible NO synthase (iNOS) (ab49999; Abcam) or β-actin (Huaan Biological Technology, Hangzhou, China). The primary antibodies were visualized with goat anti-rabbit peroxidase-conjugated antibody (Cell Signaling Technology, Danvers, MA, USA) using an enhanced chemiluminescence detection system (Millipore). The images of blots were acquired by the GBOX Chemi XT4 System (Syngene, Cambridge, UK) and GeneTools software (Syngene) was used for semi-quantitative analysis.

Immunofluorescence staining analysis was performed on 40 μm free-floating sections. Primary antibodies, including chicken anti-GFP (Aves Labs, GFP-1020), goat anti-CD206 (R&D Systems, AF2535), rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661), mouse anti-Caspase-3 (Novus Biologicals, 31A1067), mouse anti-NeuN (Millipore, MAB377), rabbit anti-Tmem119 (Abcam, ab209064), mouse anti-LPL (Abcam, ab21356), rabbit anti-CX3CR1(Abcam, ab8021), rabbit anti-GFAP (Abcam, ab7260), mouse anti-iNOS (inducible nitric oxide synthase) (Abcam, ab49999), and goat anti-Iba1 (Abcam, ab5076), were used. Corresponding secondary antibodies, including 488- and 594-conjugated secondary antibodies, were purchased from Thermo Fisher Scientific, Alexa Fluor conjugates.
All images were processed with Image J for quantification analysis. The means were calculated from 3 randomly selected microscopic fields in the ipsilateral and contralateral cortex of each section, respectively, and 3 consecutive sections were analyzed for each brain. Data are expressed as mean numbers of cells per square millimeter.

For immunohistofluorescence staining, rats were anesthetized and transcardially perfused with 200 mL 0.9% saline, followed by 400 mL 4% paraformaldehyde at seven days post-transplantation (Supplementary Fig. 3a). Thereafter, the spinal cord was dissected (1.0 cm above and below the injured site, Supplementary Fig. 3b) and cryoprotected in 30% sucrose in 0.1 M PBS dehydrate at 4 °C. The tissues were sliced at 10 mm thickness by cryostat and fixed on the PLL-coated slides. The details of immunohistofluorescence staining were consistent with the above-mentioned description (Fan et al. 2019 (link)). The primary antibodies included anti-p75 (ab245134, 1:200, Abcam), GFP (ab6673,1:200, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:200, Abcam), Arg-1 (ab60176, 1:200, Abcam), NF68 (2835s, 1:50, Cell Signaling Technology) and DAPI (ab228549, 1:1000, Abcam). Other following procedures were same as the immunocytofluorescence.