Mouse monoclonal anti βiii tubulin

Immunostaining for GFAP and β-III-tubulin was performed as previously described^{29 (link)}. The primary antibodies used for this were: mouse monoclonal anti-βIII-tubulin (1:1000, Cell Signaling Technology, Boston, MA, USA) and rabbit polyclonal anti-GFAP (1:3000, Dako, Hamburg, Germany). The secondary antibodies used were: donkey anti-rabbit IgG labeled with AlexaFluor® 488 (1:1000) and donkey anti-mouse IgG labeled with AlexaFluor® 594 (1:1000), from Invitrogen (Carlsbad, CA, USA). Samples were coded before immunostaining and blinded quantifications of labeled cells were done in nine independent experiments. Experiments were performed and quantified by different individuals to avoid subjective bias during the quantification process. Original fluorescence colors have been changed in some of the pictures shown, using the ImageJ software, to enhance visualization of markers.

HCT116 cells were seeded and allowed to adhere overnight. The cells were treated with Dulbecco's modified Eagle's medium (DMEM) with or without 125 μg ml⁻¹ or 15 μg ml⁻¹ rOly. Cells were fixed in 3.7% (v/v) formaldehyde in phosphate-buffered saline (PBS) with 0.5% (v/v) Triton X-100, blocked in PBS containing 5% (v/v) donkey serum (Jackson, New Baltimore, PA, USA), and incubated with rabbit polyclonal anti-caveolin-1 (anti-Cav-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with mouse monoclonal anti-β-III tubulin (Cell Signaling Technology, Danvers, MA, USA) or anti-rOly specially prepared for our group by Adar Biotech Ltd. (Rehovot, Israel). Cells were incubated with Alexa Fluor® 488 (ab150077) goat anti-rabbit IgG secondary antibody (Abcam, Cambridge, UK) and phalloidin- Tetramethylrhodamine (Phalloidin-TRITC). Nuclei were stained with 1 mg ml⁻¹ 4′6-diamidino-2-phenylindole (DAPI). Stained cells were examined under an inverted light microscope (Nikon Eclipse E400) at 40X magnification.