Lsm 880 nlo

All C. elegans (N2 and VC1024) were obtained from the Caenorhabditis Genetics Center (University of Minnesota, MN, USA). N2 (wild-type C. elegans) and VC1024 (parkin-null C. elegans) were maintained and reproduced at 20 °C in C. elegans growth medium (NGM) with E. coli uracil strain OP50 seeds as a nutriment. To eliminate any effects caused by the difference between C. elegans ages, each experiment was carried out in the synchronous C. elegans population. N2 and VC1024 were cultured in common NGM medium, and the experimental group was cultured in medium containing 50 μM and 100 μM VB12, respectively. Imaging was performed using L4 C. elegans. Each group of C. elegans was immersed in an aqueous solution containing an ROS probe (DCFH-DA) for 5 min, then crawled on an OP50-free NGM for 30 min, and finally transferred to a slide coated with 2% agarose gel. Imaging was carried out with a Zeiss LSM880 NLO (2 + 1 with BIG). The study on the movement distance of C. elegans was selected as a behavioral experiment. At least 30 C. elegans were removed from each group, and the movement distance of each C. elegans in OP50 free medium in 1 min was calculated. In this study, the movement distance of C. elegans was calculated by the number of complete sinusoidal movements.

The adsorption of nanoparticles at the oil–water interface can be observed by confocal laser scanning microscopy (CLSM) (LSM 880 NLO, Carl Zeiss AG, Ober-kochen, Germany). PE was diluted by 100 times before shooting. The emulsion was further characterized with the method of Li et al. with minor modifications [32 (link)]. The oil phase was stained with Nile red, and the protein phase was stained with FITC during the emulsion preparation. Emulsions were observed under a 10× objective. The laser excitation source for Nile red was 488 nm and the laser excitation source for FITC was 543 nm.

Mat slices were mounted on a microscope slide or tissue culture plate and imaged by brightfield and confocal fluorescence microscopy, either using a Zeiss Laser Scanning Microscope (LSM) 700 confocal scanning laser system coupled with a Zeiss Axiophot inverted microscope, or a Zeiss LSM 880-NLO with ZEN software (Zeiss, Jena, Germany). Slices were initially imaged by light microscopy to gain an understanding of general mat architecture, and photomosaics were obtained. On the LSM700, fluorescence imaging was performed using laser excitation at 488 and 555 nm, with a Plan-Neofluar 10 × 0.3 NA and C-Apochromat 40x (NA 1.2) water immersion objective, collecting the emitted fluorescence between 300–550 and 578–800 nm, respectively. On the LSM880, a 561 nm laser was used with a C-Apochromat 40x (NA 1.2) water immersion objective, with a 570–695 nm band pass filter.

CAFs-cancer cells cocultures were imaged using a two-photon laser-scanning microscope (Zeiss LSM880NLO) in single photon mode, using a 40×/1.30 OIL DICII PL APO (UV) VIS-IR objective, and laser lanes 488 and 561. CAF rings were ablated using a Ti:Sapphire laser (Mai Tai DeepSee, Spectra Physics) set at 800 nm and laser power of 10% Image acquisition was started 10 s before the ablation, every 2 s and for a total time of 50 s.
To perform laser ablations in vivo, living tumor tissue slices from control or myosinIIA KO mice were prepared as described previously^{10 ,40 (link)}, and placed into a 35 mm glass-bottom culture dish. A slice anchor (SHD-26GH/10; Harvard Apparatus) was placed on top of the tissue slices to minimize sample drift and covered with a drop (100 µl) of DMEM-GlutaMAX (Glibco), supplemented with 1% (v/v) antibiotic-antimycotic (Gibco), 2.5% (v/v) fetal bovine serum, 1% (v/v) Insulin-Transferrin-Selenium (ITS, ThermoFisher Scientific) and 10 ng/mL EGF (Peprotech). The ablation setup was adapted from the in vitro experiments, with laser power set at 20%. Image acquisition was started 10 s before the ablation, every 2 s and for a total time of 50 s. Para-Nitroblebbistatin (50 µM) (Optopharma) was added to 4–5 slices per mouse, incubated at 37 °C, 5% CO₂ for 2.5 h, and then ablations were performed as described.

Nuclear and cytoplasmic fractions were isolated using the NORGEN kit (Cat. 21000, NORGEN, USA). Briefly, indicated cells were lysed using Cell Fraction Buffer on ice for 10 min. Subsequently, after centrifugation at 5000 g for 5 min at 4 °C, the supernatant or the pellet were collected for further cytoplasmic or nuclear fraction purification, respectively. For RNA fluorescence in situ hybridization (FISH) assay, Cy3-labelled PKMYT1AR probe was designed and synthesized by RiboBio (China), and the FISH kit (RiboBio, Fluorescent In Situ Hybridization Kit, Cat. C10910) was used to detect the non-coding RNA expression pattern following the manufacturer’s instructions. 4,6-diamidino-2-phenylindole (DAPI) was used to indicate nuclear. All images were obtained with an LSM880 NLO (Zeiss) confocal microscope system.

We followed a previously reported immunofluorescence analysis protocol to study the hnRNP A1 distribution (30 (link), 54 (link)). In brief, wild-type and Hsp27-KO RD cells were infected by EV-A71 at an MOI of 20 or 40 for 6 h. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with 0.3% Triton X-100 in PBS. The cells were blocked with 5% BSA in PBS for 2 h and stained by incubation with anti-hnRNP A1 antibody (antibody sc-32301) and then with Alexa Fluor 488-conjugated anti-mouse immunoglobulin antibody. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) for 5 min. The images were captured on a Zeiss laser scanning microscope (LSM 880 NLO with Airyscan; Germany).

The fresh roots of P. heteroclada and P. nigra were sampled after 15 days of waterlogging stress treatment. The maturation zone of the roots was subjected to a HitoCore NANOCUT R-automatic rotary microtome (Leica, Berlin, Germany) for sectioning. The image of the root tissues was observed through lignin autofluorescence excited by an excitation wavelength of 498 nm by using a confocal laser microscope (LSM 880 NLO, Zeiss, Oberkochen, Germany). In addition, the root imaging data of P. heteroclada and P. nigra under CK and WS treatments were imported into ZEISS ZEN Lite (blue edition) software for the histological observation and calculation of the aerenchyma in the air cavity area of the roots (sample root air cavity area = total area of section air cavity/total area of section surface).