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Fig. 1. Clustering of GM1 in laminin-1-treated DRG cells. (A) Immunostaining of endogenous GM1. DRG cells were treated with or without 5 µg/ml of laminin-1 in the presence of 100 ng/ml NGF. Cells were stained using anti-GM1 antibody (left) or CTxB (right) and analyzed by confocal laser microscopy. Bars, 5 µm. Arrowheads indicate clustering of GM1. (B) BODIPY-GM1 staining. Micrographs show DRG cells labeled with 500 nM BODIPY-GM1 (green) and then incubated with DiI-C18 (red). DRG cells were analyzed by confocal laser microscopy 5 minutes after the addition of 5 µg/ml of laminin-1. Arrowheads indicate clustering of BODIPY-GM1. Bar, 5 µm. Graphs show fluorescence intensities of BODIPY-GM1 (in arbitrary units; green curve) and DiI-C18 (in arbitrary units; red curve) versus the length of indicated lines within the area (indicated 1 and 2; in µm) of the two merged micrographs 5 minutes after addition of laminin-1. DiI-C18 fluorescence intensity was measured using Leica LCS software. Laminin-1 induced focal the aggregation of BODIPY-GM1 in the membrane, but not of DiI-C18 – which labeled the membrane in general. (C) Analysis of endogenous GM1 by immunogold electron microscopy. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were then fixed and stained with anti-GM1 antibody and secondary antibody conjugated to 5-nm gold particles to detect GM1. Labeled cells were analyzed by electron microscopy. Boxed areas 1 and 2 for NGF alone and NGF+laminin-1 treatment in the left panels are shown enlarged as the middle and right panels. Treatment with NGF alone showed no clustering of gold particles to detect GM1 (arrows). Treatment with NGF and laminin-1 together induced clustering of gold particles to detect GM1 (encircled areas).
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