Lsm 880 laser confocal microscope

To confirm that M1-EVs and M2-EVs were internalized by GBM cells, EVs were stained and labeled with Dil (Beyotime Biotechnology) following the manual and visualized with an LSM880 laser confocal microscope (Zeiss, Cambridge, UK). They were ultracentrifuged at 100,000×g for 60 min to remove residual dye and resuspended. Then, U87MG cells were cocultured with Dil-labeled EVs for 12 h. After refreshing the culture medium, Alexa Fluor™ 488 phalloidin (Invitrogen, Waltham, USA) and Hoechst (Beyotime Biotechnology) were added to label cells and EV internalization was observed after washing.

Embryos were selected around 30 hpf, anesthetized using 0.04 mg/mL MS222 (Sigma, St. Louis, MO, USA) and mounted in 1% low-melting point agarose (Sigma, St. Louis, MO, USA) over glass bottom dishes. After agarose gelification and during image acquisition, embryos were kept in Ringer’s solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.2) with 0.04 mg/mL MS222. Live acquisitions were made using a Zeiss LSM 880 laser confocal microscope, with a 40×, 1.2 NA silicone oil immersion objective. Stacks around 40 µm-thick were acquired in bidirectional scanning mode, at 1 µm spacing and 512 × 512 pixel resolution every 15 min, for 2.5–16.5 h. The acquisition time per embryo was approximately 1 min, and up to 8 embryos were imaged in each experiment. The embryos were fixed in 4% PFA immediately after the end of the time-lapse, and processed for further confocal microscopy, labeling nuclei with methyl green.

The coding sequence of Sg2204 (without a signal peptide) was constructed in pCAMBIA1300 with TagGFP fused at the C‐terminus. All constructs, including the empty vector pCAMBIA1300, were transformed into Agrobacterium tumefaciens GV3101 and cultured overnight in LB medium (50 μg/mL kanamycin, 25 μg/mL rifampicin) at 28 °C. The cells were harvested and resuspended in infiltration medium (10 mM MgCl₂, 10 mM 2‐(N‐morpholino) ethanesulphonic acid, 200 μM acetosyringone, pH 5.6) to an OD600 of 0.5. Agrobacterium carrying the effector‐GFP constructs were then infiltrated into the leaves of 6‐week‐old N. benthamiana plants using a 1‐mL syringe without a syringe needle. The infiltrated leaves were collected at 3 days after infiltration and observed under a Zeiss LSM 880 laser confocal microscope (Zeiss, Jena, Germany). Cell nuclei were stained with 4,6‐diamidino‐2‐phenylindole (DAPI). GFP was excited at 488 nm, and DAPI signals were analysed at 405 nm.

Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/. 15 March 2023) and WoLF PSORT II (https://wolfpsort.hgc.jp/. 15 March 2023) were used to predict the subcellular localization of ZcTPS02.
The pCAMBIA 1300-35S-sGFP vector was digested with the restriction enzymes Sac I and Xba I. The ZcTPS02 gene was then inserted into the pCAMBIA 1300-35S-sGFP vector. After sequencing to confirm successful insertion, the vector was transferred to Agrobacterium GV3101 and cultured on plates containing kanamycin and rifampicin for 2–3 days. The Agrobacterium containing ZcTPS02-GFP and the Agrobacterium containing the P19 gene were incubated at 28 °C and 200 rpm until they reached OD₆₀₀ = 0.5–0.6, respectively. After centrifugation at 5000 rpm for 10 min, both were resuspended in infiltration buffer containing 0.5 mM MES (pH 5.6), 0.2 mM acetosyringone (AS), and 1 mM MgCl₂, adjusted to OD₆₀₀ = 1.0, mixed in equal volumes, and left at room temperature for 2–3 h. The epidermis of the tobacco leaves was infiltrated and the plants were grown under normal lighting for 36–48 h after an 8-h dark period. The GFP fluorescence signal was observed using a Zeiss LSM 880 laser confocal microscope [35 (link)].

Phenylthiourea-treated embryos were fixed at 48 hpf or 5 dpf as described above. They were then immobilized on glass slides using 2% agarose and injected with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Thermo Fisher Scientific, Waltham, MA, USA) or 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO; Thermo Fisher Scientific, Waltham, MA, USA) dissolved in chloroform. For optic chiasm observation in 48 hpf embryos, DiI was injected into the vitreous chamber of one eye, whereas DiO was injected into the contralateral eye. For optic tectum observation in 5 dpf larvae, DiI was injected into the vitreous chamber of one eye. In all of the cases, after the injection the embryos or larvae were incubated for 48 h at room temperature and the dissected brains were mounted in 1.5% agarose-50% glycerol in 20 mM Tris buffer (pH 8.0) and stored at 4 °C or −20 °C. Observation was performed using a Zeiss LSM 880 laser confocal microscope, with a 25 × 0.8 NA glycerol immersion objective.

HaVAP1, HaVAP1^‐sp, HaVAP2, HaVAP2^‐sp and HvCLP were constructed in pYBA1137 with TagRFP fused at the C‐terminus. All constructs, including empty vector pYBA1137, were transformed into A. tumefaciens EHA105 and cultured overnight. After precipitation, Agrobacterium cells were suspended in infiltration buffer containing 100 μm acetosyringone (Sigma‐Aldrich, St. Louis, MO, USA), 10 mm 2‐(N‐morpholino)ethanesulfonic acid (Sigma‐Aldrich) and 10 mm MgCl₂ to reach an optical density at 600 nm (OD₆₀₀) of 1.5. The Agrobacterium suspensions were kept at room temperature for 3–5 h and were then infiltrated into N. benthamiana leaves with a 1‐mL syringe. Three days after infiltration, the infiltrated leaves were collected and observed under a Zeiss LSM 880 laser confocal microscope (Zeiss, Jena, Germany). Plasmolysis and DAPI markers were performed by infiltrating a 30% glycerine–water solution and 1 μg/mL DAPI in N. benthamiana leaves, respectively. RFP, chlorophyll autofluorescence and DAPI signals were excited at 561, 488 and 405 nm, respectively, and collected at 586–647, 680–700 and 410–556 nm, respectively.

The intracellular ROS generation was detected using a ROS assay kit (Abcam) according to the manufacturer's instructions. Briefly, adherent NPC cells, tumour single cell suspensions, or tumour tissue sections of NPC patients were incubated with DCFH‐DA probe at 1:1000 dilution in serum‐free RPMI medium at 37°C for 30 min, then washed with serum‐free RPMI medium three times and visualized under a LSM880 laser confocal microscope (Zeiss) or detected by a fluorescent reader (TECAN) with excitation at 488 nm.