Mouse anti lamin b1

Cells were grown on 12 mm glass coverslips (Electron Microscopy Sciences, Hatfield, PA) in complete medium. Cells were fixed and permeabilized using 4% PFA and 0.1% Triton X-100 using standard methods. Cells were blocked for 30 minutes in 1% BSA. For nuclear size determination, cells were labeled in blocking buffer with 1:1000 mouse anti-Lamin B1 (AbCAM, Cambridge, MA) for 30 minutes at room temperature. Cells were stained for 30 minutes at room temperature in secondary antibody (goat anti-mouse Alexafluor488, Life Technologies). For cytoskeleton fluorescence, cells were stained with phalloidin-AlexaFluor647 (Life Technologies) and counterstained with DAPI according to the manufacturer’s instructions. ProLong Gold (Life Technologies) was used to mount the coverslips. Cells were imaged at room temperature using a Nikon Eclipse 90i upright fluorescent microscope and 10× or 20× objective (Nikon 10× PlanFluor DIC L/N1, 0.30 NA; Nikon 20× Plan Apo VC DIC N2, 0.75 NA). Images were captured using a Nikon Digital Sight DS-Qi1Mc camera at 1024×1024 or 1280×1024 pixel resolution. To quantify nuclear size, the images were thresholded using ImageJ. The “analyze particles” function was used to determine the nuclear area. At least 60 cells of each type were analyzed for nuclear size.