Ab6672

The intracisternal (i.c.) transplantation of CSF and drug administration into the medulla segment were conducted as previously described [14 (link), 15 (link)]. In brief, we utilized a custom-made syringe with a 45° angled, length-limited (4.0 mm) needle. During CSF transplantation, 15 μl of CSF from either sham or CCI-ION model rats was injected on each occasion, with a total of four injections. To neutralize IL-6 in the CCI-ION CSF prior to injection, rabbit anti-IL-6 antibody (abcam, ab6672, 0.05 μg/μL) was mixed with the CSF and incubated at 37 °C for 1 h. As a control, rabbit isotype IgG was used. The applied drugs included rabbit isotype IgG (CST, 3900, 0.25 μg/μL), anti-IL-6 antibody (abcam, ab6672, 0.1 μg/μL and 0.25 μg/μL), and IL-6 protein (1.0 μg and 5.0 μg), with each injection volume adjusted to 5.0 μL.
For CSF collection, rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.). A skin incision was made above the epencephalon to expose the foramen magnum. CSF were carefully drawn out using an insulin needle, appearing as a yellowish transparent liquid. Collected CSF samples were immediately flash-frozen with dry ice and subsequently stored at − 80 °C until required for analysis or application.

A nuclear extraction kit (Ab110168, Abcam, Cambridge, MA, USA) was used for the preparation of nuclear extracts from sciatic nerve tissues. For Western blotting, frozen rat sciatic nerve tissues were homogenized and the lysates were prepared in ice-cold lysis buffer. Nuclear extracts or total protein were collected and normalized for equal amounts of total protein measured by the bicinchoninic acid (BCA) method. Seventy micrograms of protein from each sample was separated on a sodium dodecyl sulfate polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated overnight with primary anti-NF-κB antibody (1:1000; sc-109, Santa Cruz, CA, USA), anti-IL-6 antibody (1:1000; ab6672, Abcam, Cambridge, MA, USA), anti-TNF-α antibody (1:1000; AB1837P; Millipore, Billerica, MA, USA), and anti-β-actin (1:5000, Sigma, St. Louis, MO, USA) at 4 °C, followed by incubation with the suitable HRP-conjugated secondary antibody for 4 h. β-actin protein was immunodetected as the internal standard.