Sc 13914

For brain histology studies, rats (d60) were deeply anaesthetized with pentobarbital (100 mg/kg) and fixed by transcardial perfusion with 4 % paraformaldehyde. Total brains were removed and placed in 4 % paraformaldehyde solution overnight at 4 °C. Samples were next placed in serial dilutions until fixed in 100 % ethanol and embedded in paraffin. Coronal sections were cut into 8–10 μm sections and immunohistochemically stained with goat polyclonal anti-MBP antibody (sc-13914, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution in blocking buffer and donkey anti-goat secondary antibody (sc-2020, Santa Cruz, Biotechnology, Santa Cruz, CA) at 1:500 in 1 % BSA. The staining was developed with DAB substrate (Vector Laboratories, Burlingame, pt]?>CA) and sections were counterstained with toluidine (0.1 %) blue. Images were acquired under microscope at 40X magnification (DP Olympus BX51). Areas of MBP fibers were assessed as MPB-positive per high power field and quantified using ImageJ software (NIH, Bethesda, MD).

OEG and hippocampal cell cultures were grown on 13 mm coverslips and fixed for 15 minutes in paraformaldehyde (4%) in phosphate buffer saline (PBS). Coverslips were washed three times with PBS and blocked for 30 min with 1% horse serum + 2% bovine serum albumin (BSA) in PBS with Triton 0.25% (suppressed in O4 staining). The blocking solution was then removed and the primary antibodies of interest were added [P75 (1:100) (#D4B3, Cell Signaling), CB1 (1:100) (SAB2500190, Sigma-Aldrich), MAGL (1:100) (ab152002, Abcam), CNPase (1:100) (SAB4200693, Sigma-Aldrich), Olig2 (1:200) (ab81093, Abcam), O4 (1:300) (produced in hybridoma), MBP (1:100) (sc-13914, Santa Cruz Biotechnology), β-III-tubulin (1:500) (GTX50789, GeneTex)] for overnight incubation at 4°C. Coverslips were washed with PBS and fluorochrome-conjugated secondary antibodies donkey anti-goat Alexa 488, goat anti-mouse Alexa 488 and/or donkey anti-rabbit Alexa 594 (1:400) (Alexa Fluor, Life Technologies) were added for 2h at room temperature protected from light. After rinsing with PBS, coverslips were mounted using a PBS 40% glycerol and DAPI 0.04 μg/ml solution and then examined in a fluorescence microscope (ApoTome 2^® - ZEISS).