Article Title: Nogo-A Modulates the Synaptic Excitation of Hippocampal Neurons in a Ca2+-Dependent Manner
Figure Lengend Snippet: Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p
Article Snippet: In the case of the AMPA receptors, the anti-AMPAR 1 GluA1 (Alomone Labs, Jerusalem, Israel, Cat# AGP-009, 1:50) and anti-AMPAR 2 GluA2 (Alomone Labs, Cat# AGC-005, 1:50) were co-applied with the Nogo-A or control antibodies for 10 min. After completion of the treatment, the coverslips were rinsed with pre-warmed NB- medium and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB containing in mM 50 NaH2 PO4 *2H2 O, 85 Na2 HPO4 *2H2 O) for 10 min at room temperature (RT).
Techniques: Immunolabeling, Immunofluorescence, Marker, Blocking Assay, Fluorescence