Anti inos

Animals were anesthetized with a lethal dose of pentobarbital, and the brains were perfused via the ascending aorta with ice-cold phosphate-buffered saline followed by cold 4% paraformaldehyde. Tissues were deep-frozen on dry ice, serial 10 μm coronal sections were prepared, and immune-fluorescent stainings were performed as previously described (30). The following antibodies were used: mouse anti-NeuN (1:200, Chemicon), rabbit anti-Iba1 (1:220, Wako), anti-iNOS (1:1000, Novus biologicals), anti-β amyloid (1:800, BioLegend). Goat anti-rabbit Alexa-fluor 488 (1:200, Invitrogen), goat anti-rat Alexa-fluor 488 (1:200, Invitrogen), and goat anti-mouse Alexa-fluor 555 (1:200, Invitrogen) were used as secondary antibodies where appropriate. Nuclear counterstain was performed using DAPI (Vector Laboratories).

Animals were anesthetized with pentobarbital and perfused with ice-cold PBS followed by cold 4% paraformaldehyde. Spinal cords were placed in OCT and deep frozen in dry ice. Serial 10 μm longitudinal sections of spinal cords were prepared, and immunofluorescence stainings were performed as previously described (59 (link)). The following antibodies were used: anti-CD3 (Bio-Rad Laboratories, MCA1477), anti-IBA1 (Fujifilm Wako, 019-19741), anti-iNOS (Novus Biologicals, NBP2-22119), anti-NEUN (MilliporeSigma, MAB377), anti-APC (Ab-7, Calbiochem, OP80), anti-MDA (Abcam, ab6463), anti-GFAP (Dako, Z0334), anti-NG2 (MilliporeSigma, AB5320), anti-MPB (MilliporeSigma, MAB386), and anti-NF M (Chemicon International, AB1987). Goat anti–rabbit Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11034), goat anti–mouse Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11001), goat anti–rabbit Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A27039), goat anti–rat Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A21434), and goat anti–mouse Alexa Fluor 555 (1:200, Invitrogen, Thermo Fisher Scientific, A28180) were used as secondary antibodies appropriately. Nuclear counterstain was performed using DAPI (Vector Laboratories). Bielschowsky silver impregnation (for axons) and Gold-Black (for myelin) staining were performed as previously described (60 (link)).

The protocol has been described previously [12 (link)]. Proteins of interest were detected using anti-iNOS (1:1000, Novus), anti-eNOS (1:1000, Abcam), anti-alkaline phosphatase (ALP; 1:1000, Abcam), anti-runt-related transcription factor 2 (Runx2; 1:1000, Abcam), anti-peroxisome proliferator-activated receptor (PPAR)γ (1:1000, Abcam), anti-p-JNK (1:1000, Abcam), or anti-JNK (1:1000, Abcam) antibodies, while β-actin was detected with anti-β-actin antibody (1:2000, Abcam) as a control.