Deltavision rt deconvolution microscope

Human dermal fibroblasts were grown on cover slips in a 6-well plate a day before the experiment. The following day, the cells were fixed and permeabilized by incubating them with methanol (–20°C) for 20 minutes. Cells were washed 3 times for 5 minutes with phosphate-buffered saline (PBS) and then incubated with primary antibody against lamin A/C (E-1, or lamin B1 [L1220-20, mouse monoclonal antibody, US Biological; www.usbio.net]) [25 ] at a dilution of 1:100 for 60 minutes at 37°C in a humidified chamber. Cells were then washed 3 times for 5 minutes with PBS and incubated with AlexaFluor-568–coupled fluorescent secondary antibody (Invitrogen) [26 ] for 60 minutes at 37°C in a humidified chamber. After incubation, cells were washed 3 times for 5 minutes with PBS, counterstained with 4’-6-diamidino-2-phenylindole (DAPI) during the washes, and mounted on a glass slide with Aqua Poly/Mount (Polysciences). Cells were observed with DeltaVision RT Deconvolution Microscope (Applied Precision). Z-stack images for red and blue fluorescence were acquired in 0.15-µm steps at 60× magnification and were deconvolved using SoftWoRx (Applied Precision). Images were further processed using Imaris software (Bitplane).