The left sciatic nerve was exposed at mid-thigh level. A crush injury was induced to the sciatic nerve for 15 sec, using a pair of microforceps. After injury, the site of crush was marked
with sterilized ink. Then the overlying muscle and skin were sutured, and the animals were returned to warm cages to recover from anesthesia. All experimental protocols were approved by the local ethics committee and in accordance with the U.K. Home Office regulations (Animals Act 1986). Perfusion and tissue processing At three, 14, 28, or 56 days postsurgery (n= 4 per time point), animals were overdosed Inhibitors,research,lifescience,medical with sodium pentobarbital (60 mg/kg; Sagatal, France) and perfused via the ascending aorta with 4% paraformaldehyde Inhibitors,research,lifescience,medical in 0.1 M phosphate buffer, pH 7.4. A piece of the left sciatic nerve was dissected
out, spanning from at least 5 mm proximal to the crush site to about 10 mm distal to the site, where the sciatic nerve starts to branch. The tissue was postfixed in the same fixative for 2 h at room temperature (RT), and then cryoprotected in 30% sucrose solution in 0.01 M phosphate buffered saline (PBS) overnight at 4°C. All sciatic nerves were then embedded in OCT medium (BDH Laboratory Supplies, Poole, UK) at –20°C and stored at –80°C until further processing. Inhibitors,research,lifescience,medical Four additional control rats, which had sham operations, were also processed in this way. Serial longitudinal sections of 8-μm thickness were cut using a cryostat, and consecutive sections processed for immunohistochemistry. Antibodies and immunohistochemistry The following primary
antibodies were Inhibitors,research,lifescience,medical used: mouse anti-200-kDa neurofilament (an axonal marker; N52 clone, Sigma, Gillingham, Dorset, UK; 1:1000), mouse anti-P0 (a peripheral myelin marker; Inhibitors,research,lifescience,medical clone 18 against aa32–38 of P0; Astexx, Austria; 1:3000), mouse anti-S100 (a Schwann cell marker; Sigma, UK; 1:2000), mouse antimyelin-associated glycoprotein (MAG, a marker for incisures; Chemicon, Temecula, CA; 1:200), mouse anti-pan NaV channels (a nodal marker; Sigma, UK; 1:50), rabbit anti-JAM-C polyclonal anti-body (1:800~1:1500). Lectin staining was performed using the fluorescein-labeled jackfruit agglutinin (jacalin, a nodal marker; Vector, Servion, Switzerland; 1:100). Appropriate secondary antibodies conjugated why with Alexa Fluor dyes were purchased from Invitrogen (Grand Island, NY, USA) as follows: goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 586 (all at 1:400). Hoeschst 33342 (Sigma, UK; 0.2 g/100 mL PBS) was used to reveal cell nuclei. All primary and secondary antibodies as well as the PTC124 mw normal goat serum (NGS) were diluted in PBS containing 0.2% Triton X-100 and 0.1% sodium azide. The PBS wash (3 × 10 min) was a standard routine after incubation with a primary or secondary antibody. All incubation was carried out in a humidified chamber at RT. The general procedure for immunohistochemistry was as follows.