Biotinylated rabbit anti streptavidin

TK6 cells were cultured for 48 h with 100 nM aphidicolin and incubated with 0.1 μg/ml Colcemid for 3 h before collection. The cells were swollen in hypotonic solution (75 mM KCl) and fixed with ethanol-acetic acid (3:1). Fluorescence in situ hybridization (FISH) was carried out as previously described [42 (link)]. FRA3B was probed with BAC clones RP11-170K19 and RP11-495E23. Immunodetection was performed by alternating incubations with the following antibodies: streptavidin-Cy3 (1:200) (Invitrogen), biotinylated rabbit anti-streptavidin (1:266)(Rockland, Limerick, PA) and mouse anti-digoxygenin FITC (1:50)(Interchim, Montluçon, France) and goat anti-mouse Alexa 488 (1:200, Invitrogen) The cells were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA).

A PDMS microchip was used as a substrate for UV grafting (Sylgard 184, Dow Corning, Midland, MI, USA). Benzophenone, 2-aminoethyl methacrylate (AEMA), monoclonal mouse anti-CD63 antibody, and FITC-labeled streptavidin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biotinylated mouse anti-CD63 antibody was purchased from BioLegend (San Diego, CA, USA). Biotinylated rabbit anti-streptavidin (Rockland Immunochemicals, Gilbertsville, PA, USA) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from FUJIFILM Wako Pure Chemicals Corporation (Osaka, Japan). All other reagents were of analytical grade or higher.