Alexa 488 antibodies

Cultures of hippocampal neurons were prepared from E18 Sprague-Dawley rats (male and female). The animal procedures were approved by the ethical committee of the University of Bordeaux. Cells, from both dorsal and ventral hippocampi, were plated at a density of 50 × 10³ cells per ml on poly-lysine pre-coated cover slips. Coverslips were maintained in a 3% horse serum containing neurobasal medium. This medium is replaced after 4 days in vitro (DIV) by a serum-free neurobasal medium. Cultures were maintained at 37 °C in 5% CO₂ for 20 div at maximum. For surface NMDAR immunostaining, GluN2A-Flag and GluN2B-SEP subunits were co-transfected in neurons at 8–10 div. To image glutamatergic synapses, Homer 1c-DsRed was also co-transfected. Surface GluN2A-Flag and GluN2B-SEP subunits were stained in live neurons using antibodies against FLAG (Sigma, mouse, 1/500, 15 min, 37 °C) and SEP/GFP (R & D systems, rabbit, 1/500, 15 min, 37 °C), respectively. Neurons were then fixed (paraformaldehyde 4%) and incubated with secondary antibody directed against anti-rabbit Alexa 635 antibodies (Invitrogen, 1/500, 45 min) and anti-mouse Alexa 488 antibodies (Invitrogen, 1/500, 45 min). Neurons were washed, mounted, and preparations were kept at 4 °C until observation.

Flow cytometry analyses were conducted to analyze the surface expression of MAGEA proteins in different EVs. 10 μg of EVs from each sample were incubated with 10 μl of 4 μm diameter aldehyde/sulphate latex beads (Molecular Probes; Life Technologies) for 15 minutes at room temperature. Then PBS was added to a final volume of 1 ml and the mix was incubated at 4° C overnight using end-over-end rotation. The beads were then blocked with a 100 mM glycine/PBS solution for 30 minutes at room temperature and washed twice with 0.5% BSA in PBS. Incubation with affinity-purified rabbit polyclonal antibodies against MAGE-A4 (final concentration of 1 ng/μl) or MAGE-A10 (2 ng/μl)) [28 (link)] or mouse monoclonal antibodies 6C1 (sc-20034; Santa Cruz) and anti-E2Tag 3F12 (Icosagen) were carried out in 0.5% BSA in PBS for 1 hour at 4° C using end-over-end rotation. The samples were then washed twice. Anti-rabbit or anti-mouse Alexa 488 antibodies (1 mg/ml, dilution 1:1000, Invitrogen) were used as secondary antibodies and samples were incubated with it for 1 hour at 4° C using end-over-end rotation. The beads were washed twice, resuspended in 300 μl of 0.5% BSA in PBS and analyzed with the LSR II device (BD Biosciences) using the BD FACSDiva Software (BD Biosciences). Analysis of the results was carried out with the FlowJo VX program (Tree Star).