Notes

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However, Conn does believe that ibogaine can make a difference for certain people who are addicted to drugs. Since these are central nervous system depressants, or tranquilizers and sedatives, they are generally prescribed for treating sleep disorders and anxiety disorders. When ingested, the alkaloid interacts with different sites in the central nervous system. It takes around 4-8 weeks of time on the SAO so the level of the methadone in the system can drop sufficiently. Briefly, sections were post-fixed in 4% paraformaldehyde/PBS, acetylated in 0.1 mtriethanolamine/0.25% acetic anhydride, and permeabilized for 10 min (nervous system tissues) or 5 min (kidney, spleen) in 1% Triton X-100/PBS. Briefly, sections were permeabilized with ice-cold 95% ethanol, 5% acetic acid for 25 min and rehydrated in PBS blocked for 1 hr with PBS containing 0.1% cold-water fish gelatin (Aurion, Wageningen, The Netherlands), 2.5% normal goat serum, and 0.05% saponin. Antibodies were diluted in the same buffer without saponin. For kidney and other non-neural tissues, ethanol permeabilization has been omitted, and sections were blocked for 1 hr with PBS containing 5% normal goat serum and 0.1% saponin. For semithin sections, nerves embedded as for electron microscopy were cut in 2 μm sections and stained with alkaline toluidine blue.

Semithin sections and electron microscopy. Tissue preservation and electron microscopy. Electron microscopy of sciatic nerves of transcardially perfused experimental and control mice (2% PFA and 2% glutaraldehyde in 0.1 mcacodylate buffer) was performed according to Adlkofer et al. COS cell transfection. ibogaine uk was performed as described previously (Schaeren-Wiemers et al., 1995c). The transfected COS cells were fixed after 48 and 60 hr with 4% paraformaldehyde in PBS, permeabilized with 0.1% saponin, 0.1% Tween 20, or 0.1% Triton X-100 in PBS, blocked with 5% normal goat serum in PBS for 1 hr, and processed for immunocytochemistry as described for the kidney using FITC-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Antibodies. Synthesis of peptides and immunization of rabbits was done by Research Genetics (Huntsville, AL) using the 13 mer peptide MFDGFTYRHYHEN corresponding to amino acids 114-126 of rMAL protein as described previously (Schaeren-Wiemers et al., 1995b). Immunizations were also done in our laboratory using this peptide with the RIBI adjuvans system (RIBI Immunochemicals, Hamilton, MT), with a similar result. Primary antibodies were omitted in the controls. For double-staining experiments, primary and secondary antibodies were applied simultaneously.

Monoclonal antibodies against MBP (Boehringer Mannheim) and PLP (Boehringer Ingelheim) were used at a dilution of 1:500. The primary antibodies were incubated overnight at 4°C or for 2 hr at room temperature. Antibodies directed against PMP22 (Susi 4) (Pareek et al., 1993) or directed against the myelin protein P0 (P0raba; gift from Dr. M. Filbin, Hunter College, NY, NY) were both applied at a dilution of 1:1000 at 4°C for 16 hr. This blocking buffer was also used for all antibody dilution steps, whereas PBS was used for the washing steps. Secondary antibodies (horse radish peroxidase-coupled polyclonal goat anti-rabbit antisera; Sigma, St. Louis, MO) at a dilution of 1:5000 were applied for 1-2 hr at room temperature after fourfold washing of the membrane with PBS/0.2% Tween 20. After repeated washing to remove excess secondary antibodies, detection of the immunoreactive products was performed by visualization of chemiluminescence (ECL Western blot detection reagents; Amersham, Arlington Heights, IL) on x-ray films (Fuji, Stamford, CT). Data has been created ᠎wi th the help of GSA Con tent Generator  DE᠎MO!

Detection was performed with alkaline phosphatase-coupled anti-digoxigenin antibodies (Boehringer Mannheim) and revealed with a chemiluminescent substrate (CSPD; Tropix, Bedford, MA). Stringent washes were done in 0.2% SSC at 68°C, and hybridization signals were visualized after incubation with alkaline phosphatase-coupled anti-digoxigenin antibodies (Boehringer Mannheim) using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as a color reaction substrate. After prehybridization for 3 hr at room temperature, the sections were hybridized overnight at 68°C with sense or antisense digoxigenin-labeled probes in hybridization buffer containing 5× SSC, 50% formamide, and 2% blocking reagent (Boehringer Mannheim). Digoxigenin-labeled riboprobes were generated from pBluescript SK-vector (Stratagene, La Jolla, CA,) containing the full-length sequence of rMAL cDNA (Schaeren-Wiemers et al., 1995b) with T3 (antisense) and T7 (sense) RNA polymerase, using digoxigenin-UTP (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. Ten micrometer cross-cryosections of the sciatic nerves of 8-, 21-, and 70-d-old wild-type and PMP22-transgenic mice were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s recommendations. Light microscopic analysis. Six micrometer frozen sections from the quadriceps muscle were stained by NADH tetrazolium reductase histochemistry according to standard protocols and analyzed and photographed on an Axiophot microscope.


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