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After incubation with the antibody/complement mixture, the cells were washed twice with MEM and cultivated under neurosphere forming conditions at a clonal density of 1250 cells/ml. When immunocytolysis was performed with cells from mixed neural cultures, the latter were grown to confluence for 6 ? days and subsequently were incubated with 0.05 Trypsin/EDTA (Invitrogen) until the cells detached from the cell culture dish to obtain a single cell suspension. The cells were then diluted in MEM with 0.2 BSA, and immunocytolysis was performed as described for the acutely dissociated cells from E14.5 cortex. Following immunocytolysis, the cells were plated on poly-D-lysin (Sigma) coated cell culture dishes (Nunc, Wiesbaden, Germany) for 24 h in DMEM supplemented with 10 (v/v) horse serum before analysis. Transfection of COS7 Cells--COS7 cells were routinely cultured on uncoated 10-cm dishes at 37 , 6 CO2 in DMEM supplemented with 10 (v/v) FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. Lipofection was performed according to the manufacturer's protocol using the Lipofectamine 2000 reagent (Invitrogen). The cells were transfected with a fucosyltransferase 9 (Fut9) expression plasmid, containing full-length mouse Fut9 cloned into pcDNA3.1 or the empty pcDNA3.1 vector (Invitrogen) as a negative control. To identify transfected cells by GFP fluorescence, the cells were cotransfected with pEGFP-N1 (Invitrogen; ratio pcDNA3.1/pEGFP-N1 5:1). Immunocyto- and Immunohistochemistry--Immunocytochemical staining was performed as outlined previously (27). For staining of acutely dissociated cells, cells from embryonic tissue were resuspended in neurosphere medium and plated on Laminin-coated dishes for 2 h before immunocytochemical analysis was performed. The cells from postnatal tissue were grown in DMEM (Invitrogen) 10 FCS on Laminin for 24 h before analysis. For simultaneous staining of mAb 5750 withMAY 6, 2011 ?VOLUME 286 ?NUMBERmAbs 487LeX and MMALeX, 5750 antibody was linked to NHSPEO4-Biotin (AppliChem), and stainings were performed sequentially using streptavidin Cy3 (Dianova) conjugate. For immunohistochemistry, E13.5 or E14.5 embryos were removed, and immersion was fixed for 8 h with 4 (w/v) paraformaldehyde in PBS at 4 . Tissues were cryoprotected overnight with 20 (w/v) sucrose in PBS, embedded in tissuefreezing medium (Jung, Nussloch, Germany), and frozen on dry ice. The sections (14 ?6 m) were cut on a cryostat (Leica, Solms, Germany). Before immunostaining, cryosections were rehydrated in blocking solution (PBS/10 FCS) for 2 h at room temperature in a humidified slide chamber. Primary antibodies were diluted in blocking solution and incubated overnight at 4 . The sections were washed three times for 5 min with PBS containing 0.1 BSA (PBS/A). Secondary antibodies were diluted in PBS/A and applied for 2 h in the presence of Hoechst (1:100,000). After three final washing steps with PBS, the cryosections were mounted with glass coverslips in ImmuMount. Glycanase https://britishrestaurantawards.org/members/redcandle3/activity/360534/ Digestion--L1-CAM, Tenascin-C, and Phosphacan were purified from mouse brains as previously described (23, 24). 300 g of affinity-purified protein were adjusted to a final concentration of 0.4 (w/v) SDS and 2 (v/v) -mercaptoethanol and a volume of 40 l in PBS. The samples were boiled for 5 min at 95 . After cooling on ice, the denatured samples were diluted 1:1 with N-glycosidase F buffer (150 mM Na2HPO3 pH 8, 50 mM EDTA, 3 (v/v) Nonidet P-40) before addin.