Sab4200747

On postoperative day 30, the nerve samples (n = 6 per group) were dehydrated in alcohol, embedded in paraffin, and cut lengthwise into 4-μm-thick sections. After the sections were incubated in 3% hydrogen peroxide for 25 minutes, nonspecific sites were blocked with 3% albumin bovine serum for 30 minutes. Next, the sections were separately incubated with mixtures of phosphatebuffered saline and primary antibodies against anti-neurofilament 200 (NF-200) (1:200, SAB4200747, Sigma), anti-α-smooth muscle actin (α-SMA) (1:200, ab5694, Abcam), and anti-sigma-1 receptor protein (S1R) (1:400, 15168-1-AP, Proteintech) overnight at 4°C. Goat antimouse (ab97265, Abcam) was selected as the secondary antibody for NF-200, and goat anti-rat (ab205720, Abcam) was selected for α-SMA and S1R. Six images (×400 magnification) were randomly assessed in each rat using the immunohistochemistry (IHC) toolbox plugin 19 of ImageJ. The percentage mean positive area was used (mean positive area ratio = positive area pixels/total image area pixels × 100%).

Standard immunofluorescent procedures were used to assess the longitudinal nerve sections according to the methods of Hobson et al. 20 Anti-myelin basic protein (MBP) (1 μg/ml, ab40390, Abcam) and anti-NF-200 (5 μg/ml, SAB4200747, Sigma) were used. Six images (×200 magnification) were randomly selected with a Nikon Eclipse Ci series fluorescence microscope. Percentage positive areas for MBP and NF-200 (×100%) were calculated using ImageJ. Mean percentage positive area was used in analyses (mean positive area ratio = positive area pixels/total image area pixels × 100%).