centration of 107 cpm/ml of hybridization solution. After hybridization, hydrolysis of nonspecifically hybridized probe was initiated by treatment of brain sections with 30 mg/ml ribonuclease A in 10 mM Tris saline with 1 mM EDTA for 40 min at 458C. Subsequently, the sections were rinsed through descending concentrations of SSC buffer containing 100 mM so- dium thiosulfate. The distribution of hybridization was localized by an ex- posure to Beta Max film (Amersham Pharmacia Biotech) and Eastman Kodak Co. NTB2 emulsion. Films were exposed for 2–4 d and subjected to liquid emulsion for 2–8 wk. Tissue sections were counterstained with hematoxylin and eosin after autoradiographic development. Cell Culture Cell Line. A conditionally immortalized mouse oligodendrocyte cell line (CIMO) (Bronstein et al., 1998) was grown in DME/HAM F12 supple- mented with 5% FCS, transferrin, selenium (Reduser; Upstate Biotech- nology), and gamma interferon (Ig; 100 U/ml) (GIBCO BRL) at 338C or 378C in 5% CO2. Cells were either maintained in medium with Ig at 338C (permissive) or 378C in medium lacking Ig (nonpermissive). CIMO cells were stable-transfected with pBabe vector, OSP/claudin-11, antisense OSP/claudin-11, PLP, and MBP in pBabe vector separately and pcDNA3.1 vector alone and OAP-1 in pcDNA3.1 vector. DOTAP liposo- mal transfection reagent (Boehringer) was used for transfection according to the manufacturer’s protocol. Stable transformants were maintained af- ter antibiotic (neomycin 400 mg/ml for pBabe vector background and genetecin 500 mg/ml for pcDNA vector background) selection. Purification of Oligodendrocytes. Purified oligodendrocytes were ob- tained using a technique modified from McCarthy and de Vellis (1980). In brief, primary cultures were established from rat and mice (wild-type, OSP/claudin-11 homozygous, and heterozygote knockout) neonatal fore- brain cells obtained by dissociating cerebral cortices in papain and grown for z10 d in DME/F12 supplemented with 10% heat-inactivated FCS. The flasks were shaken overnight to separate the loosely attached oligoden- drocyte precursors. These cells were centrifuged and resuspended in ap- propriate medium and used for immunocytochemistry and cell migration assays (Milner et al., 1996). Antibodies Anti–OSP/claudin-11 antibody was made as described previously (Bron- stein et al., 1997). Rabbit polyclonal antibodies for OAP-1 were raised against an 18–amino acid peptide (H-YSDWENTDWFKETKNQSV- OH) conjugated to keyhole limpet hemocyanin (Research Genetics). The 18 amino acids (153–170) (see Fig. 1) correspond to a region within the putative second extracellular loop of OAP-1. Antibody specificity was confirmed by Western blot, immunohistochemistry, and peptide competi- tion experiments. Anti-GFAP antibody, peroxidase-conjugated, and horseradish-conjugated goat anti–rabbit antibody were purchased from Zymed Laboratories. Anti–b1 integrin antibody (Chemicon), Texas red– labeled and FITC-labeled goat anti–rabbit antibody, FITC-labeled goat anti–mouse antibody (Vector Laboratories), and anti-GalC antibody (Boehringer) were used in different concentrations for immunohis- tochemistry and in some cases for Western blots and migration assays. Immunohistochemistry and Confocal Microscopy Oligodendrocytes were grown on Lab-Tek chamber slides for 4–8 d. For experiments in fixed cells, cultures were washed with PBS, fixed with 4% paraformaldehyde for 20 min at 48C, and treated with 0.3% TX-100 in PBS plus 2% normal goat serum for 10 min at room temperature either before or after incubation of cells with primary antibodies. Some cells were also treated with 100% ice-cold methanol for 10 min at 2208C. Both live and fixed cells were blocked for 2 h in 25% normal goat serum in TBS; primary antibody was either added for 2 h at room temperature or overnight at 48C. Antibody dilutions were as follows: OSP/claudin-11, 1:50; OAP-1, 1:250; and b1 integrin, 1:50. Antigen–antibody interactions were visualized with anti–rabbit IgG conjugated to Texas red (1:3,000) and FITC (1:2,000) and anti–mouse IgG conjugated to FITC (1:1,000). Live cells were then fixed with 4% paraformaldehyde. After staining, slide chambers were re- moved, and slides were coverslipped with Fluoromount (Fisher Scientific). Cells were examined with a ZEISS LSM 410 laser confocal microscope system. The excitation source was a krypton argon laser (Coherent) with output at 488, 568, and 633 nm. Fluorescein fluorescence was imaged with a 488-nm emission filter and a 515–540-nm band pass filter. Texas red flu- orescence was imaged with a 568-nm emission filter. The resulting images were created by projecting several optical sections obtained at different 1-mm intervals through the cells in the z-axis. Immunogold Electron Microscopy Primary oligodendrocytes were grown in filter chambers (Corning) for 3 d before washing them three times with PBS. The cells were fixed in 1% glutaraldehyde and 2% paraformaldehyde for 20 min at room tempera- ture. They were permeabilized and blocked in a similar manner used for confocal immunocytochemistry. OSP/claudin-11 and OAP-1 antibodies were used at 1:50 concentration. The cells were washed and incubated with anti–rabbit IgG conjugated to 12-nm colloidal gold particles (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. They were washed and processed by standard methods, and ultrathin cryosectioning was performed as described previously (Bronstein et al., 2000). Cell Surface Biotinylation Cell surface proteins of primary oligodendrocytes were biotinylated by us- ing a method modified from Mody et al. (1999). In brief, primary oligo- dendrocyte monolayers were washed and then incubated for 30 min at room temperature in the presence of 1 mM sulfosuccinimydyl-6 (biotina- mido)-hexanoate (Sulpho-NHS-Biotin; Pierce Chemical Co.) in PBS. For immunoprecipitation, flasks were washed with PBS and 0.5 M Tris-Cl, and cells were solubilized in buffer A with 1% Triton X-100. Cells were solubi- lized after washing with PBS and 0.5 M Tris-Cl, centrifuged, and Strepavi- din-agarose beads (Sigma-Aldrich) were added to the supernatant. After 3 h of incubation, beads were collected by brief centrifugation and washed. Immunoprecipitated protein was dissociated from the beads by addition of SDS-containing sample buffer with b-mercaptoethanol and DTT and heating for 4 min. The sample was subjected through 12% SDS- PAGE, and electroblotted protein was probed with anti–OSP/claudin-11 and anti–OAP-1 antibodies. Immunoprecipitation and Western Blot Analysis Primary oligodendrocytes, CIMO cells, and biotinylated primary oligo- dendrocytes grown to confluence were washed with 13 PBS. Primary cells and mouse brain homogenates were homogenized in PBS, 0.6 mM PMSF, 1.5 mM EDTA, 0.003% leupeptin, and 0.6 mM DTT. The brain and cell homogenates were subsequently resuspended in buffer A (20 mM Tris- HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100) for 30 min on ice. Insoluble material was pelleted by centrifugation at 18,000 g for 10 min at 48C. Cel- lular lysate was precleared with 40 ml of protein A (Sepharose/agarose) beads for 1 h at 48C and centrifuged at 3,000 g for 10 min. Specific mole- cules were immunoprecipitated by the incubation of 120-mg protein super- natant with purified primary antibody overnight at 48C, followed by incu- bation with protein A beads for 1 h at 48C. The beads were washed three times for 15 min with cold buffer A and resuspended in SDS-PAGE load- ing buffer. 30 mg of cell homogenate per lane was subjected to SDS- PAGE on 12% (wt/vol) acrylamide gels (Laemmli, 1970). Proteins were transferred to nitrocellulose membranes by semidry electroblotting. Blots were blocked in 5% BSA in TBS-T (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20 [vol/vol]) and then probed with primary antibodies for either 2 h at room temperature or overnight at 48C. The blots were washed with TBS-T and probed with HRP-conjugated secondary anti- body. Chemiluminescence substrate (Pierce Chemical Co.) was used for the HRP reaction. Proliferation Assays To evaluate the effect of OSP/claudin-11 and OAP-1 overexpression on proliferation, [3H]thymidine incorporation was measured in CIMO cells stably transfected with either pcDNA3.1 vector alone, pcDNA3.1-contain- ing OAP-1, pBabe vector alone, or pBabe-containing OSP/claudin-11, an- tisense OSP/claudin-11, PLP, and MBP. Overexpression of OSP/claudin- 11, OAP-1, and controls was confirmed by Northern and Western blot analysis. Cells were incubated with [3H]thymidine (0.2 mCi/well) for 16 h under permissive or nonpermissive conditions, washed, and DNA was pre- cipitated with 5% TCA and subsequently determined by a liquid scintilla- tion counter. Incorporated radioactivity was measured in triplicates. All experiments were performed in triplicates and done at least four times. Cell Migration Assay Cell migration was quantified by measuring the number of cells migrating from the agarose drops using a modification of the method described by Varani et al. (1978). Oligodendrocytes were resuspended at 4 3 107 cells/ ml in Sato medium containing 10% heat-inactivated FCS and 0.3% low melting agarose maintained at 378C to prevent setting of the agarose. Tiwari-Woodruff et al. OSP/Claudin-11, b1 Integrin, and OAP-1 Form a Complex 297