Tcs sp8 wll

haESCs were incubated with 0.4 mg/mL demecolcine (Sigma) for 1 h. After trypsinization, the cells were resuspended in 0.075 M KCl at 37 °C for 15 min and then fixed in methanol: acetic acid (3:1 in volume) for 30 min. The cells were dropped onto pre-cold and precleaned slides.
For karyotype analysis, the protease-treated cells were stained with Giemsa dye (Yeasen) for 15 min. Pictures were taken by Olympus BX53 and more than 50 metaphase spreads were analyzed. The G-banded ideogram of chromosome images was arranged according to the previous publication ^{56 (link)}.
For cell FISH experiments, whole chromosome probes XMP15 and XMP17 were hybridized following the manufacturer’s protocol (MetaSystems), and nuclei were counterstained with DAPI. Pictures of the chromosomes were acquired by using Leica TCS SP8 WLL. Only the stained chromosomes were analyzed.

Seven days after CCI, the mice were sacrificed, and their spinal cords were removed and postfixed in 4% paraformaldehyde (PFA) overnight at 4°C. After dehydration, the tissues were paraffin embedded and sectioned (7 μM) on a microtome (Leica, RM45). Adjacent coronal sections from corresponding regions of the lumbar (L4 to L6) spinal cords of naive and CCI mice were incubated overnight at 4°C with the following primary antibodies: rabbit anti-XCL1 (1:50, Novus Biologicals), rabbit anti-ITGA9 (1:50, Abcam), rabbit anti-XCR1 (1:50, Lifespan Biosciences), mouse anti-NeuN (1:250, Merck), rat anti-IBA1 (1:1000, Abcam), and chicken anti-GFAP (1:10000, Merck). Antigen-bound primary antibodies were visualized with appropriate Alexa Fluor 488/594–conjugated donkey secondary antibodies (1:100, Invitrogen). Hoechst 33342 (Invitrogen) was used to stain cell nuclei. Stained sections were examined and acquired under a high-class confocal microscope (Leica TCS SP8 WLL) equipped with HyD, PMT and TLD detectors. The ipsilateral part of the lumbar spinal cord was visualized on representative images.

Cells grown on glass slide for 2 h were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 20 min and then in 0.1 M HCl for 5 min. The cells were then washed with 2× SSC for 5 min twice and then washed in 50% formamide/4× SSC for 10 h at 4 °C. The hybridization was the same as mentioned above.
The images were acquired on Leica TCS SP8 WLL. For each imaging view, z-stacks covering the whole nuclei with a step size of 400 nm were taken for each channel and imaging conditions were kept for different views of one sample. DAPI was stained to represent the nuclear profile. The 3D image analysis was carried out in Imaris (Bitplane) by ImarisCell, a module designed specifically to identify, segment, track, measure and analyze cell, nucleus and vesicles in 3D images. For 3D chromosome FISH image analysis, “Surface” function was used to segment nuclear boundary by DAPI channel and chromosome territory boundary of Chr15 and Chr17 by 488 nm and 552 nm channel intensity, respectively. The volume and center of mass of nucleus and chromosome territories were output directly. The volume of each nucleus was measured to normalize the volume of chromosome territories. Distance between nuclear center of mass and chromosome territories was normalized by the cubic root of nuclear volume. We only selected haploid cells for chromosome FISH analyses.

All the plates were scanned with the automated microscope for well-plates, Image Xpress Nano Automated Imaging System (Molecular Devices, LLC, San Jose, CA, USA), with three fluorescent filters (dopaminergic neurons) or TCS SP8 WLL (Leica, Wetzlar, Germany) in widefield mode (cortical and hippocampal neurons). Acquired images of each well were analyzed with the CellProfiler, and the CellProfiler Analyst software packages [46 (link)] adapted as previously described by us [38 (link)].

Confocal section images were captured using Leica DM6000 TCS/SP8 laser confocal scanning microscope. Confocal whole-mount images were captured using Leica TCS SP8 WLL.