Journal: PLoS ONE

Article Title: Site-specific HNK-1 epitope on alternatively spliced fibronectin type-III repeats in tenascin-C promotes neurite outgrowth of hippocampal neurons through contactin-1

doi: 10.1371/journal.pone.0210193

Figure Lengend Snippet: Primary hippocampal neurons were cultured on PDL, PDL coated with FNx7 without HNK-1 expression (HNK-1(-)), or PDL coated with FNx7 with HNK-1 expression (HNK-1(+)). Normal goat IgG (+ normal IgG) or anti-CNTN pAb (+ CNTN Ab) was added to the culture medium at a final concentration of 30 μg/mL after 18 h of culture. Neurons were immunostained and analyzed at DIV3. Green, PDL with normal goat IgG (n = 219); light green, PDL with anti-CNTN pAb (n = 130); yellow, FNx7 HNK-1(-) with normal goat IgG (n = 139); light yellow, FNx7 HNK-1(-) with anti-CNTN pAb (n = 163); orange, FNx7 HNK-1(+) with normal goat IgG (n = 154); and light orange, FNx7 HNK-1(+) with anti-CNTN pAb (n = 166). Numbers of measured neurons were indicated in parentheses. This experiment was performed across two independent cultures, respectively, and the representative result obtained from a single culture was shown. Error bars represent SEM. ***p < 0.001; n.s., p > 0.05.

Article Snippet: At day 3 in vitro (DIV3), hippocampal primary neurons were fixed in methanol at -20°C for 20 min and incubated with mouse anti-Tau-1 monoclonal antibody (mAb) (5 μg/mL, MAB3420 [AB_94855]; Millipore, Stafford, VA, USA) in PBS containing 3% BSA for 1 h at room temperature, followed by incubation with Alexa Fluor 546-conjugated donkey anti-mouse IgG antibody (5 μg/mL, A10036 [AB_2534012]; Molecular Probes, Eugene, OR, USA) in PBS containing 3% BSA at room temperature for 1 h. For the application of antibodies in culture medium, 30 μg/mL control goat IgG (sc-2028 [AB_737167]; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-contactin-1 polyclonal antibody (pAb) were added after 18 h of culture.

Techniques: Cell Culture, Expressing, Concentration Assay