Vectashield mounting medium

For immunocytochemistry, the cells were split onto cover slips (Cat. No. 12546, Fisher Scientific, Waltham MA) that were coated with Poly-D-Lysine (Cat. No. P8920, Sigma Aldrich, St. Louis, MO) the day before transfection. At 24 and 48 hours, the cells were fixed in 4% paraformaldehyde for 10 minutes. Following a brief 1× PBS wash, the fixed cells were permeabilized in ice-cold methanol for 5 minutes. After another wash in PBS-T, a 20% normal goat serum solution in PBS-T was used as means to block the cells for 30 minutes at room temperature. The cells were then incubated for 24 hours at 4°C in the primary antibody, C4F6 (Cat. No. MM-0070-2, Medimabs, Quebec, CA, purchased 2014), which was diluted in a 10% goat serum/PBS-T solution. The cells were washed in PBS-T prior to the addition of goat anti-mouse AlexaFlour-568 (Cat. No. A11004, Invitrogen/ThermoFisher, purchased 2014) secondary antibody and DAPI reagent in dilutions of 1:2000 each in a solution of 10% normal goat serum /PBS-T. The cells were finally washed with PBS and mounted onto glass slides (Cat. No. 1255015, Thermo/Fisher, purchased 2014) using Vectashield mounting medium (Cat. No. H1200, Vector Laboratories, purchased 2014). Pictures were taken at 20× magnification on an Olympus BX60 epifluorescence microscope.

Cells were grown on coverslips to ∼50% confluence and transfected with the respective plasmid. Forty-eight hours posttransfection, the cells were fixed with methanol and washed with PBS repeatedly. Cells were made permeable using 0.2% Triton X-100 in PBS for 15 min at 4°C and then washed with PBS. Cells were then incubated with the respective primary and secondary antibody at 4°C in a humified chamber sequentially. The coverslips were then washed and stained with DAPI (4′,6-diamidino-2-phenylindole) and mounted on slides using Vectashield mounting medium (ThermoFisher catalog number NC9265087). Images were taken using a Zeiss LSM 700 confocal laser-scanning microscope and analyzed using ZEN lite software (48 (link)).

For the quantification of micronuclei, cells were treated with 2 μg ml⁻¹ Cytochalasin B on poly-L-lysine-coated coverslips (BD Bioscience) for 12 h before being fixed and mounted with Vectashield mounting medium with DAPI (Vector laboratories). Micronuclei in binucleated cells were scored using the micronucleus assay21 (link). For the quantification of chromatin bridges and binucleation, cells were harvested following asynchronous growth incubated on Polysine (Thermo Scientific) slides, fixed and mounted with Vectashield mounting medium with DAPI. Slides were scored using a Nikon Ellipse Microscope. Binucleation was defined and scored as in the micronucleus assay.

Whole-mount tissues were stained according to our previously published methods^{47 (link)–49 (link)}. Briefly, tissues were fixed at 4 °C with 4% PFA in PBS overnight, washed with PBS, and cut into small pieces. Tissues were digested with proteinase K (20 mM) and permeabilized with 100% methanol. Samples were blocked overnight with a PBS-0.1% Triton X-100 solution containing 3% milk, and incubated at 4 °C overnight with primary antibodies against CD31 (1:100, Cat. No. AF3628, R&D) and Perilipin (1:200, Cat. No. 20R-PP004, Fitzgerald Industries). After washing with PBS, samples were blocked with 3% milk, and incubated at RT with fluorescent-conjugated secondary antibodies (1:300, Thermo Fisher Scientific Inc.) for 2 h. After rigorous rinsing, samples were mounted with a Vectashield mounting medium (Cat. No. H-1000, Vector Laboratories). Fluorescent signals were examined under a confocal microscope (LSM510 Confocal, Zeiss).