Alexa 647 goat anti mouse antibodies

Multi-plexed Western blots were performed by transferring proteins to Imobilon-FL membrane in Running Buffer (0.025M Tris, 0.192 M Glycine with 20% methanol, the blot was blocked in TBS (20mM Tris pH 7.6, 143 mM NaCl) plus 0.2% Tween-20 and 5% nonfat dry milk. Primary antibodies (anti-GlActin [14 (link)], anti-acetylated tubulin 6-11-B1 (Sigma), or anti-HA HA7 (Sigma)) were incubated overnight at 1:3000 at 4°C. After three washes (5, 10, 15 minutes) the blot was incubated for 60 minutes with secondary goat anti-rabbit-HRP (Bio-Rad, 1:7000) and Alexa 647 goat-anti-mouse antibodies (Molecular Probes, 1:2500). After three washes with TBS-T the blot was imaged with a Chemidoc MP (Bio-Rad). Values were normalized against the indicated loading controls after measuring band intensity with the ImageJ multi-measure function.

Cells were processed for immunocytochemistry as previously described^{69 (link)}. Briefly, NS grown on glass were previously fixed 15 minutes at room temperature in 4% paraformaldehyde and incubated at 37 °C for 1 h with primary antibodies directed against ki67 (1/200, rabbit, Abcam), β-III-tubulin (1/400, TuJ-1 clone; rabbit; Abcam), MAP-2 (1/200, mouse; Sigma), CNPase (rabbit, Covance) and GFAP (1/500, mouse; Sigma). After several rinses in PBS, samples were then incubated with Alexa-488 goat anti-rabbit and Alexa-647 goat anti-mouse antibodies (1/500, Molecular Probes) for 45 min at 37 °C. Staining of nuclei was performed using 4′,6-diamidino-2-phenylindole (DAPI, 1/500). Finally images were acquired in a LSM710 laser scanning spectral confocal microscope (Zeiss). Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio.