Lsm510 meta nlo laser scanning confocal microscope

Neurons grown on coverslips were fixed in 4% paraformaldehyde, 4% sucrose in PBS for 10 min at room temperature, and blocked in 5% BSA with 0.3 M glycine in PBST (PBS with 0.5% Tween-20) for 45 min. Primary antibodies were diluted in PBS containing 1% BSA as follows: 1:200 for goat anti-TfR1; 1:500 for rabbit anti-SDR2, goat anti-Steap2, and rabbit anti-Zip14; and 1:1000 for mouse anti-microtubule-associated protein 2 (MAP2 Santa Cruz Biotechnology, sc-80013), rabbit anti-Zip8 and rabbit anti-DMT1 and then incubated with the cells at 4°C overnight. Alexa Fluor 647-conjugated donkey anti-rabbit or anti-goat IgG, and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Life Technologies) were diluted 1:1000 and incubated with samples in the dark for 1 h at room temperature. After washing, samples were mounted onto slides with a drop of SlowFade^® Gold Antifade Reagent containing DAPI (Invitrogen). The slides were visualized using either a LSM-510 Meta NLO laser scanning confocal microscope or Axioimager epifluorescence microscope (Zeiss). The images were processed in ImageJ and co-localization analyses were performed using JACoP plugins.