Dfc420c camera

Bright‐field AO time‐lapse videos were recorded at 37°C and 5% CO₂ on an AF7000 microscope equipped with a DFC420C camera using LAS AF software (all Leica). Bright‐field cilia movement in organoids and air–liquid interface cultures was recorded using the same set‐up equipped with a Hamamatsu C9300‐221 high‐speed CCD camera (Hamamatsu Photonics) at 150 frames per second using Hokawo 2.1 imaging software (Hamamatsu Photonics).

Polyclonal mouse antibody raised against non-structural protein (NS1) of TiPV was prepared by ABclonal (Wuhan, China) and used as the primary antibody. IFA was performed according to the protocol described by Ma et al. [37 (link)] with slight modifications. Uninfected tissues samples and TiPV infected tissue samples were taken as negative controls and positive controls, respectively. These samples were fixed in 4% paraformaldehyde/PBS for 24 hours at 4°C, and washed with DPBS. The samples were cut at -20°C with cryostat and permeabilized with 0.25% TritonX-100, then blocked for 1 h at room temperature in DPBS. After washing three times with PBS, the samples were incubated with mouse anti-TiPV serum (1: 200) for 1 h at 37 º C, washed and incubated with Cy3-conugated secondary antibody (1:500, Beyotime Inc., PRC) for 1 h at room temperature. Samples were rinsed in PBS and incubated in 0.1 ug/mL 4',6-Diamidino-2-Phenylindole (DAPI) (1 ug/mL, Sigma, USA) for 5 min. Fluorescent images were captured using a Leica DM2500 fluorescent microscope with a Leica DFC420C camera at a magnification of 40x objective.

Mycelia were recovered from fungal cultures after fifteen days of incubation (50 mL; inoculated and incubated as described above, triplicate samples), mounted on glass slides and stained with lactophenol blue to enhance contrast. Visualisation of cultures was performed using a DM5500 B microscope (Leica) with 40× or 63× magnification objectives and images were captured with a DFC420 C camera (Leica).

Stereomicroscope images were acquired using a Leica MZ16F fluorescence microscope with a Leica DFC420C camera and Leica Microsystems software. Confocal images were acquired using a Zeiss LSM 780 confocal microscope and Zen 2009 software or a Leica SP8 confocal microscope and Leica Application Suite X software. Images shown in Figure 5, C and D, were acquired as 4 × 2 tile scans with a HCX PL APO CS ×10/0.40 dry objective. Images in Figure 6 were acquired using a Fluotar VISIR ×25/0.95 water objective. Images in Supplemental Figure 4A were taken with a HC PL APO CS2 63×/1.30 GLYC objective; images in Supplemental Figure 4B (P6 mesentery) were taken using a C-Aphochromat ×40/1.20 W Korr M27 objective, and images in Supplemental Figure 4, B (E18 mesentery), C, and D, were acquired using a Plan-Apochromat ×20/0.8 Ph2. All confocal images represent maximum intensity projections of z-stacks except images in Figure 6.

To produce reporter constructs and expression plasmids, PCR was carried out using specific primer pairs and N2 genomic DNA or cDNA as templates. The amplified DNA fragment was cloned into a suitable vector using restriction enzymes. The plasmids, vectors, and primers used in these experiments are shown in a supplementary data (Table S1). Each plasmid concentration for microinjection is 30 to 50 µg/ml, and total mixture concentration was adjusted to 150 µg/ml, including transgenic markers such as 50 µg/ml pRF4, 5 µg/ml pCFJ90, and 50 µg/ml unc-119 (+). The stable lines were selected at F2 generation and used for further experiments. Integration lines including jgIs40 and jgIs42 were generated by UV irradiation. Each integration line was outcrossed with wild-type worms 4 times to exclude extra mutations. For DIC and fluorescence imaging, worms were prepared on 2% agarose slides with M9 buffer and 2 mM levamisole (Sigma, L9756, St. Louis, MO, USA). Photographs of worms were captured using a DFC420C camera (Leica) attached to an Axio Imager A1 microscope (Zeiss).
The intensity of the fluorescent protein was measured using the ImageJ program (https://imagej.net/). The measurements of the marked area were achieved by the plot profile and displayed graphically after background subtraction.

Cells (3 × 10⁵) were washed in 1x PBS and spun onto a glass slide using a Shandon Cytospin 4 (Thermofisher), fixed and stained with the Diff-Quick stain kit (Dade Behring S.A., Brussels, Belgium) according to the manufacturer's procedure. Images were acquired using a Leica DM2000 equipped with a DFC420C camera and Leica FireCam software.