Dmi8 confocal laser scanning microscope

GUS histochemical staining was performed following the procedure described by [57 (link)]. Rice tissues were immersed in X-Gluc (Thermo Fisher Scientific, USA) staining solution at 37 °C overnight and were subsequently rinsed in 70% ethanol at room temperature for 1 or 3 days. Pictures were taken with an Olympus SZX12 stereo microscope. The transfected rice protoplasts were incubated at room temperature for 16–20 h. Fluorescence microscopy was carried out on a Leica DMi8 Laser Scanning Confocal microscope (Leica, Germany) with Excitation/emission wavelengths 488/535 nm for green fluorescence.

Intestinal tissue slides were deparaffinized with xylene and rehydrated with an alcohol gradient. To enhance immunoreactivity, the slides were incubated in 10 mM sodium citrate for 15 min at 95°C. Then the slides were cooled to room temperature, washed in PBS for 5 min (five times in total), blocked for 2 h with 5% Bovine Serum Albumin (BSA), and incubated for 2 h with Ulex europaeus agglutinin-1 (UEA-1). Finally, 4′,6-diamidino-2-phenylindole(DAPI)was used to counterstain nuclei. For lysozyme (Lyz) staining, cells were stained with anti-rabbit lysozyme antibody (1:200, Abcam) overnight at 4°C. The samples were incubated with goat anti-rabbit to Alexa Fluor 594 (1:250, Abcam) for 90 min, followed by DAPI for 5 min at room temperature. For in vitro imaging, infected organoids were embedded in Matrigel on glass chamber slides. The 0.5% Triton X-100 was used for 20 min to permeabilize the cells. Thereafter, the slides were washed with PBS three times and incubated for 1 h in 5% BSA. Subsequently, UEA-1 and Lyz were used to visualize goblet cells and Paneth cells in organoids, respectively, and the staining was observed with a Leica DMi8 Laser Scanning confocal microscope (Leica, Germany).

Colocalization of organellar markers was done by imaging with a Leica DMi8 Confocal Laser Scanning Microscope. Fluorescence images were captured at 20 °C by an EMCCD camera (iXon888, Andor Instruments) using a ×60 objective (N.A. = 1.40; water) and suitable filter sets for EGFP and mCherry (ex. 488 and 561, respectively).

The terminal transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostic Corporation. For detection of apoptosis, TUNEL assay was performed following the manufacturer’s instructions. Briefly, the fixation process was performed as described above. After fixation, the samples were permeabilized with 0.1% Triton X-100 in PBS for 30 min at 37 °C. The TUNEL reaction was then performed using the manufacturer’s instructions. Hoechst33342 (5 mg mL⁻¹ in PBS, Gibco) was used to visualize the nuclei. The state of cell clusters stained by the TUNEL was detected by a Lecia DMI8 confocal laser scanning microscope. Images of five to six randomly chosen fields of vision (one-two spheroids per field) were taken.

Confocal microscopy images were acquired using a Leica DMI8 confocal laser scanning microscope with excitation wavelength at 488 nm. Optical sections were obtained every 5 µm steps inside sweat channel to access to plug 3D structure. Image J software was used to process the captured images.