Tcs sp8 laser confocal microscope

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).

The root tips of 2-week-old transgenic tobacco (pROKII-PsnCYCD1;1-GFP) seedlings were collected and flatten on a glass slide for microscopic observation. The wild-type and transgenic tobacco with a pROKII-GFP vector was used as control. The green fluorescent signals in cells were observed and photographed by Leica TCS SP8 laser confocal microscope (Leica, Wetzlar, Germany).

90 µl of paraformaldehyde fixed cells was incubated with 5 × 10⁻⁷ M DAPI and 1:100 Vybrant™ DiD Cell-Labeling Solution (Invitrogen) at 37°C for 20 min. Samples were centrifuged at 2000 g × 1 min. They were washed with 1 ml PBS and sprun at 2000RPM x 3 min twice. Finally, they were resuspended in 80 µl PBS then dropped on cavity slides (Eisco), covered with coverslips (FisherScientific), and analyzed with Leica TCS SP8 Laser confocal microscope. Visualization of neutrophils was performed with water immersion objective HC PL APO CS2 40x/1.10. Images were obtained in sequential scanning mode using Diode 405, OPSL 488, OPSL 552 and Diode 638 lasers. We used scan speed 200 Hz and pixel size 0.223 µm for flat images. Z-stacks were obtained with scan speed 600 Hz, XY pixel size 0.223 µm and voxel size 0.424 µm. Images and Z-stacks were processed with LASX (Leica microsystems). Images and Z-stacks were converted to TIFF images for analysis in ImageJ (FIJI distribution 2.1.0/1.53q). Processing and analysis, including image thresholding, fluorescence colocalization, and per-cell analysis of fluorescence signals, employed custom ImageJ macros, code for which is provided in full in the supplement. For per-cell analyses, regions of interest were drawn manually around each imaged cell, using images of DiD membrane stain to define the edges of individual cells.