Journal: Frontiers in Synaptic Neuroscience
Article Title: N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells
Figure Lengend Snippet: Analysis of N-cadherin-mediated effect on synaptic vesicle accumulation in cholinergic neurons. CHO cells transfected with EGFP, N-cadherin-EGFP, or N-cadherin-EGFP and p120-catenin-EGFP were plated on 5 days old brainstem cholinergic nuclei explants and cocultured for 2 days. Tissue cultures were fixed and immunostained with SV2 (A) or synapsin I (B) antibodies. (A,B) Confocal images of CHO cells transfected with EGFP (a,b,c) , N-cadherin-EGFP (d,e,f) , or N-cadherin-EGFP and p120-catenin-EGFP (g,h,i) . Panels (a) , (d) , and (g) , show SV2 or synapsin I immunostaining (red); panels (b) , (e) , and (h) , show merged images with EGFP fluorescence (green); and panel (c) , (f) , and (i) , show EGFP fluorescence alone. A white dashed line delineates the contour of the transfected CHO cell in each panel. (C) N-cadherin expression levels in CHO cells transfected with N-cadherin or cotransfected with N-cadherin and p120-catenin were measured as pixels of N-cadherin immunolabeling over CHO cell surface area (μm 2 ) (N-cadherin, n = 31 cells; N-cadherin and p120-catenin, n = 27 cells). (D) Analysis of (a) SV2 immunolabeled area (μm 2 ) over transfected CHO cells normalized to CHO cell surface area (μm 2 ), and (b) SV2 average gray value [arbitrary units (AU)] over transfected CHO cells normalized to CHO cell surface area (μm 2 ) [EGFP ( n = 18), N-cadherin ( n = 24), and N-cadherin and p120-catenin ( n = 24)]. (E) Analysis of (a) synapsin I immunolabeled area (pixels) normalized to neurite length (μm) in contact with transfected CHO cells, and (b) synapsin I total gray value (AU) per neurite length (μm) in contact with transfected CHO cells [EGFP ( n = 10), N-cadherin ( n = 14), and N-cadherin and p120-catenin ( n = 12)]. (C) Student's T -test, * p
Article Snippet: To detect N-cadherin and p120-catenin, cells were fixed in 4% paraformaldehyde, blocked and permeabilized with 5% donkey serum containing 0.2% Triton X-100 (Sigma) in D-PBS, and incubated with anti N-cadherin cytoplasmic domain mouse monoclonal antibodies (clone 32/N-cadherin) (BD Bioscience), washed, and incubated with an anti mouse IgG Cy3-conjugated secondary antibody (Jackson ImmunoResearch). p120-catenin was immunodetected with a mouse monoclonal antibody (Invitrogen, 6H11).
Techniques: Transfection, Immunostaining, Fluorescence, Expressing, Immunolabeling