Tcs sp8 sted 3 microscope

The stained cells were imaged using a Leica TCS SP8 microscope at lower magnification, while higher resolution imaging with subsequent deconvolution (Huygens Professional) was performed on a Leica TCS SP8 STED 3× microscope (Leica Microsystems, Germany).

Stimulated emission depletion microscopy (STED) was performed on a Leica TCS SP8 STED 3× microscope, Leica Microsystems (Wetzlar, Germany). Samples were irradiated with a pulsed white light laser at wavelengths 499 and 590 nm. A continuous wave STED laser line at a wavelength of 592 nm, Leica Microsystems, was used for depletion of the 488 nm fluorophore, reaching a lateral resolution of ~70 nm. The signal was detected using a gated hybrid detector (HyD), Leica Microsystems, in photon‐counting mode. STED images were acquired using a dedicated oil objective with 100× magnification and a numerical aperture of 1.4 (Leica Microsystems). A Z‐stack was made with a step size of 150 nm and pixel size of 19 × 19 nm, optimized using Nyquist Calculator from SVI (Scientific Volume Imaging, Hilversum, the Netherlands). Finally, deconvolution was performed with Huygens Professional (SVI).

The DoDELLA1 gene fragment without a stop codon was amplified by PCR then inserted into the EcoRI site of the pSAT6-EYFP-N1 expression vector to construct a YFP-fusion vector [115 (link)]. A. thaliana mesophyll protoplasts, which are useful for subcellular localization experiments, were used as the transient gene expression system. Protoplasts were isolated from the mesophyll of leaves of four-week-old A. thaliana plants [116 (link)]. Using a PEG-mediated method [116 (link)], recombinant plasmids (YFP-DoDELLA1) and NLS localization marker (NLS-mCherry) were co-transformed into protoplasts. A Leica TCS SP8 STED 3 × microscope (Leica Microsystems, Wetzlar, Germany) was used to detect YFP and NLS fluorescence signals in protoplasts after incubation in the dark for 12–18 h. Supplementary Table S2 lists the primer pairs that were used to generate the YFP-DoDELLA1 fusion protein.

The 4T1 cells (0.1 × 10⁶/mL) were plated in an 8-well Nunc™ Lab-Tek™ II Chambered Coverglass dish (Thermo Scientific™, Waltham, MA, USA) with a No. 1.5 borosilicate glass bottom. In their exponential phase of growth, cells were incubated for 24 h in media with a 4.5 mM Gd³⁺ con-centration of mannan-based polymers. After the incubation period, cells were washed twice with Hank’s balanced salt solution (HBSS, Biosera, Nuaille, France), with fluorescent dyes added in concentrations according to the producer’s manual (60–70 nM for LysoTracker^® Green and 1 μg/mL for Hoechst 33342). Incubation times were 60 min for LysoTracker^® Green and 20 min for Hoechst 33,342 [48 ,49 ]. All fluorescent dyes were purchased from Invitrogen™ by Life-Technologies (Prague, Czech Republic). After incubation, the cells were washed twice with HBSS followed by the addition of RPMI 1640 medium without phenol red. Cells were then measured under a TCS SP8 STED 3× microscope (Leica, Chicago, IL, USA; objective: HC PL APO CS2 100×/1.40 OIL). Images were displayed with automatically enhanced contrast and adjusted for brightness using ImageJ (version 1.46r, National Institutes of Health, Bethesda, MD, USA).

The ORF sequence of DoGES1 was cloned into pSAT6-EYFP-N1 at the NcoI site, which was driven by the CaMV 35S promoter. The recombinant DoGES1-YFP plasmid was transformed into 4-week-old A. thaliana protoplasts that were isolated from rosette leaves by PEG-mediated transformation as described previously [46 (link)]. After incubation at 22 °C for 14 h in darkness, YFP fluorescence signals were excited at 514 nm and with an emission wavelength of 527 nm using a Leica TCS SP8 STED 3× microscope (Wetzlar, Hesse, Germany).