Sp8 confocal microscopy

Paraffin liver sections (5 µm in thickness) were deparaffinized and rehydrated, and treated antigen retrieval (6 min, 96 °C). For histopathology, liver sections were stained with hematoxylin and eosin (H&E) (Solarbio, G1120). Images were observed and captured with a Nikon Eclipse Ni light microscopy (JAPAN). For TUNEL staining, paraffin liver sections were performed using a commercially available in situ apoptosis detection kit (Roche Molecular Biochemicals) according to the manufacturer’s protocol. And the images were visualized and captured with a Leica SP8 confocal microscopy.

TEM images were acquired on a Hitachi (Japan) TEM‐HT7700 at 100 kV accelerating voltage. The UV–vis‐NIR spectra were measured on a Shimadzu UV‐2600 spectrometer. DLS study was carried out on a Nano‐ZS Zetasizer (Malvern, U.K.). Fluorescence spectra were measured on the Hitachi F‐4600 fluorescence spectrometer. In vivo NIR‐II fluorescence imaging was conducted on a Series III 900/1700‐D NIR‐II imaging system (Yingrui, Suzhou). In vivo photoacoustic imaging was acquired using a commercial ORPAM system (NIR‐VIS‐50, PAOMTek Inc.). Flow cytometry analysis for cell apoptosis was performed on a FACSCanto Analyzer. The 808 and 660 nm laser was purchased from Changchun Laser Technology Co., Ltd (Changchun, China) and the infrared thermal images were recorded by FLIR E6 thermal imagers. Fluorescence confocal imaging was conducted on a Leica SP8 confocal microscopy.

Nuclear import assay was performed as described previously [53 (link)]. DLD1 cells (~0.2 × 10⁶) were seeded on coverslips in a six-well plate. After 24 h of siRNA transfection, cells were transfected with the vector encoding GR2-GFP2-M9core fusion protein. After 48 h of transfection, cells were treated with 5 μM dexamethasone (Sigma) for 30 min at 37 °C and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100 and immunostained with Phalloidin-Alexa 594 (Invitrogen, cat. A12381) to mark the cell boundary. Finally, cells were stained with DAPI and mounted in antifade solution. Images were acquired using Leica SP8 confocal microscopy using a 63× objective/N.A. 1.4 using 405, 488 and 594 nm and lasers, zoom set to 2.0. The mean fluorescence intensity of the GFP signal was determined for each cell (demarcated by phalloidin) and nucleus (demarcated by DAPI), and expressed as a ratio of the nuclear to cytoplasmic fluorescence intensity using ImageJ. Nuclear/cytoplasmic (N/C) fluorescence intensity ratios were calculated and plotted using GraphPad Prism software. Statistical analysis was performed using Mann–Whitney test. The GR2-GFP2-M9 construct was a kind gift from Ralph Kehlenbach.

To accurately count cell numbers, every 10th slide from anterior to posterior of the substantia nigra tissues was used to stain TH, NeuN, Iba1 and GFAP, respectively. The five different regions every slide were taken using 10x magnification (objective) under the Leica SP8 confocal microscopy, respectively (Fig. 4A). The five regions were fixed to all monkeys including wild-type, control group and 7, 8-DHF group. About 20 slides were used to count positive cells every monkey. Morphologically intact cell with a nucleus was counted. All of cell count results were converted into cell number/mm². Quantification data were represented as mean ± standard deviation (s.t.d) using Microsoft Excel STDEV Function. The significance difference between two samples was evaluated by the unpaired two-sample Student’s t-test using Excel software. P < 0.05 was considered as statistical significant differences.

U2OS cells were grown on poly-L-lysine coated coverslips and treated with control or CCDC66 siRNA. After 48 h of transfection, cells were treated with 5 μm/ml STLC for 16 h. Next day, cells were treated with 5 μg/ml nocodazole for 1 at 37°C. Cells were washed extensively with cold PBS to prevent MT polymerization then incubated with warm media and fixed at indicated time points and stained for alpha-tubulin and centrin. Images were acquired with Leica SP8 Confocal microscopy at 1,024 × 1,024 format with 2× zoom factor. To quantify MT nucleation area (aster size), polygon selection tool on ImageJ is used. Centrosomal and noncentrosomal MT nucleation points were defined based on centrin staining, which marks the centrioles.

HEK 293T cells transfected with the desired plasmids were fixed with 4% paraformaldehyde (Biosharp, BL539A) for 10 min at 4°C and the nuclei were counterstained with DAPI (Merck, D5492) for 15 min at room temperature in the dark, and washed three times with phosphate buffered saline (PBS). The cells were scanned and images were collected using a Leica SP8 confocal microscopy (63× oil objective NA 1.35).