Hcx pl apo 100 1.4 0 7 oil objective

The cells were arrested in mitosis by overnight incubation with 0.1 μg/ml KaryoMAX colcemid (Life Technologies). They were collected by mitotic shake‐off and swollen in hypotonic buffer (0.03 M of sodium citrate) at 37°C for 25 min. Next, the cells were fixed in freshly‐prepared 3:1 mix of methanol:glacial acetic acid and nuclear preparations were dropped onto slides pre‐soaked in 45% of acetic acid prior to being allowed to dry overnight. The following day, mitotic chromosomes were stained using Giemsa (VWR) and viewed with a Leica DMI6000B inverted microscope equipped with a HCX PL APO 100×/1.4–0.7 oil objective.

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).

Cells synchronised in mitosis via overnight incubation with 0.1 μg/ml KaryoMAX® Colcemid (Life Technologies) were collected by mitotic shake‐off and swollen in hypotonic buffer (0.03 M sodium citrate) at 37°C for 25 min. Cells were fixed in freshly prepared 3:1 mix of methanol: glacial acetic acid, and nuclear preparations were dropped onto slides pre‐soaked in 45% acetic acid prior to being allowed to dry overnight. The following day, mitotic chromosomes were stained using Giemsa (VWR) and viewed with a Leica DMI6000B inverted microscope equipped with a HCX PL APO 100×/1.4–0.7 oil objective.