Immunofluorescence Imaging of Fixed and Live Cells

Immunofluorescence was performed by combining the methods described in previous researches.¹⁰ (link)^,45 (link) Briefly, the fixed cells were permeated by 0.5% PBST (Triton X-100) and blocked in 1% BSA, followed by incubating with first and second antibodies. A DM6 microscope (Leica Microsystems) or a TCS SP8 laser confocal microscope (Leica Microsystems) was used for imaging. For confocal microscopy, 63×, 1.4 NA oil objective lenses (Leica Microsystems) was used, and the cells were imaged by scanning optical sections at ∼0.5-μm intervals. 3D reconstruction was according to confocal microscopy which scaned cell from the top to the bottom of cells based on DAPI fluorescence at ∼0.3-μm intervals, and reconstructed by LAS X software. For live-cell imaging, cells stably expressed mCherry-H2B were plated on a glass-bottom dish and first synchronized to prometaphase followed by 1 h of live photograph. Images were acquired by TCS SP8 laser confocal microscope (Leica Microsystems).