Wga alexa 555

HEK293 cells (105) were grown on sterile cover slides placed in 6-well plates. After the adhesion of the cells overnight, the cells were incubated with various concentrations of MLNCs for 24 h. Then, the cells were stained with membrane tracker WGA-Alexa555 and Nuclear Green fluorescent dyes (Molecular Probe, Eugene, USA). The cells were then washed, fixed with 4% paraformaldehyde for about 15 min, washed again, and polymerized with Mowiol 4.88 medium (Calbiochem, Nottingham, UK). Hoechst 33,342 (Sigma, USA) was used to visualize nuclei. Slides were analyzed using an Eclipse TE2000 confocal microscope (Nikon, Tokyo, Japan) at a wavelength of 405 nm.

Primary cortical cultures from E17-E18 Long Evans rat embryos were transfected with Lipofectamine 2000 (Invitrogen 11668019) at in vitro day 17 with pFUGW-eGFP. We complied with all the ethics regulations and disclose that the organization, IACUC, has approved the protocol. At in vitro day 24, cells were incubated with 1:250 anti-GluA2 (Sigma Millipore AB397) at 37 °C for 15 min. After 5 min of anti-GluA2 incubation, WGA-Alexa 555 (Invitrogen W32464) was added for 10 min. After the total 15 min incubation, cells were washed once with warm artificial cerebrospinal fluid. Cells were fixed with 4% paraformaldehyde/2% sucrose/0.000375% glutaraldehyde for 8 min at 37 °C. Cells were washed once with artificial cerebrospinal fluid and then treated with 0.001% NaBH₄ on ice for 15 min. Cells were washed three times with artificial cerebrospinal fluid and then incubated with 1:400 anti-IgG2a-Atto647N antibody (Rockland 610-156-041) in 1% ovalbumin/0.2% cold water fish gelatin blocking buffer at RT for 1 h. Cells were washed three times with artificial cerebrospinal fluid, and the dish was filled with ~5 mL artificial cerebrospinal fluid for imaging and storage.