Mice were transcardially perfused using 4% paraformaldehyde (PFA)/PBS (pH 7.4). Spinal cords were isolated, post-fixed, and dehydrated in 30% sucrose at 4°. After meninges removal, coronal sections (25μm) were prepared with a cryostat (Leica).
Spinal cord sections were incubated in warm (80°) sodium citrate solution (pH 6.0) for 20min for antigen retrieval. Samples were blocked in 3% BSA in PBS-T (0.3% TritionX-100/PBS) and then incubated with primary antibodies (rabbit anti-Iba1:500, Wako; mouse anti-nestin 1:200, Developmental Studies Hybridoma bank (DSHB); anti-GFAP 1:500, BD Biosciences; anti-Dcx, 1:200, DSHB; anti-Olig2 1:200, DSHB; anti-CD45, 1:1000, BD systems, anti-MBP, 1:200, AbDSerotec, anti-Ki67 1:500, Abcam; anti-CC1, 1:250, Abcam; anti-NG2, 1:250, Millipore) overnight at 4°C. Incubation with fluorescence-conjugated secondary antibodies at 1:1000 was performed at room temperature for 1hour, followed by washing with PBS and mounting using Fluoromount-G with DAPI (Southern Biotech). 4–8 spinal cord sections in the lumbar region were imaged per biological replicate. ImageJ (NIH) was used to quantify the intensity of fluorescent signals.
Spinal cord sections were incubated in warm (80°) sodium citrate solution (pH 6.0) for 20min for antigen retrieval. Samples were blocked in 3% BSA in PBS-T (0.3% TritionX-100/PBS) and then incubated with primary antibodies (rabbit anti-Iba1:500, Wako; mouse anti-nestin 1:200, Developmental Studies Hybridoma bank (DSHB); anti-GFAP 1:500, BD Biosciences; anti-Dcx, 1:200, DSHB; anti-Olig2 1:200, DSHB; anti-CD45, 1:1000, BD systems, anti-MBP, 1:200, AbDSerotec, anti-Ki67 1:500, Abcam; anti-CC1, 1:250, Abcam; anti-NG2, 1:250, Millipore) overnight at 4°C. Incubation with fluorescence-conjugated secondary antibodies at 1:1000 was performed at room temperature for 1hour, followed by washing with PBS and mounting using Fluoromount-G with DAPI (Southern Biotech). 4–8 spinal cord sections in the lumbar region were imaged per biological replicate. ImageJ (NIH) was used to quantify the intensity of fluorescent signals.