Photo flo

Cells grown on coverslips were washed in PBS then swollen in hypotonic solution (85.5 mM NaCl and 5 mM MgCl₂ pH 7) for 5 min. 4% paraformaldehyde fixation was performed for 10 min at room temperature and then cells were permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, X100) in PBS. After blocking in antibody diluting buffer (ADB; 1% goat serum, 0.3% BSA, 0.005% Triton X-100 in PBS) for 30 min, cells were incubated at room temperature with primary antibody diluted in ADB for 2 h. After three washes (ADB/0.4% Photo-Flo, ADB/0.005% Triton X-100, dH₂O/0.4% Photo-Flo [Kodak, 1464510]) coverslips were incubated for 1 h at room temperature with fluorochrome-conjugated secondary antibodies (1:1000, Molecular Probes, Invitrogen). Coverslips were washed twice for 5 min (ADB/0.4% Photoflo then 0.005% Triton X-100 in PBS) then dried and mounted on microscope slides using the ProLong Gold Antifade Mountant with 40,60-diamidino-2-phenylindole (DAPI; Life Technologies, P36935). Samples were viewed with a Leica DMI6000B inverted microscope and fluorescence imaging workstation equipped with HCX PL APO ×100/1.4–0.7 oil objective. Images were analysed using ImageJ software (National Healthcare Institute, USA).

C-bands (AT-rich bands) were induced through a modification of a technique used by Fernández et al. [16 (link)], which basically involves heat denaturation of chromosomal DNA in the presence of formamide, followed by incubation in 2× SSC (saline-sodium citrate buffer) at room temperature. Chromosome slides were dehydrated for 30 s in an ethanol series of 70, 80 and 98%, and kept at 65 °C for 48 h before further use. Each slide was then coated with 20 µl of 50% formamide in 2× SSC, enclosed with a cover slip, denatured for 2 min at 70 °C, and incubated at 37 °C for 1 h. After incubation, the slide was rinsed in 2× SSC for 30 to 60 min at room temperature.
The chromosome preparation was then stained with 0.5 µg/ml DAPI (4′,6-diamino-2-phenylindole; Sigma-Aldrich, Gillingham, UK) in PBS containing 1% Triton X-100 for 5 min, washed at room temperature in 1% Kodak-PhotoFlo (Kodak Alaris Inc., Rochester, NY, USA) in PBS and in 1% Kodak-PhotoFlo in miliQ water for 4 and 1 min, respectively. Finally, the slides were mounted in 25 µl of antifade based on DABCO (1,4-iazabicyclo[2.2.2.]octane; Sigma-Aldrich, Gillingham, UK).

Male and female fixed spindles slides were washed in PBS containing 0.4%Kodak Photo flo (Kodak) for 5 min, followed by 0.1% PBS-Triton X and blocked in 1XPBS-antibody dilution buffer (ADB) before being incubated over night at 4°C with the primary antibody. Primary antibodies used were: rabbit anti-ADD1 (GTX101600, Genetex. Dilution 1:100), rabbit anti-MYO10 (24565-1-AP, Proteintech. Dilution 1:100) and rabbit anti-β-tubulin (T8328, Sigma. Dilution 1:500). After overnight incubation, slides were washed to remove the unbound antibodies and incubate for 2 hours at 37°C with Alexafluor^™ secondary antibodies (Molecular Probes Eugene OR, USA). Slides were washed and mounted with Prolong Gold antifade (Molecular Probes). Image acquisition was performed using a Zeiss Imager Z1 microscope under 20X, 40X or 63X magnifying objectives, at room temperature. Images were processed using ZEN 2 (Carl Zeiss).