Airyscan lsm800

Microtubule regrowth experiments were performed as described previously (61 (link)). Briefly, microtubule regrowth after tubulin depolymerization in cold shock was analyzed. WFA-treated or untreated LFA-1/ICAM-1-stimulated T-cells were washed and fixed after cold shock recovery at 37 °C for 15 s and processed for AiryScan confocal microscopy (Zeiss LSM800 AiryScan, Carl Zeiss).

Seeds were sterilized with 10% NaClO for 15 min and then washed with ddH₂O 5–6 times to remove the NaClO. Surface‐sterilized cucumber/wheat seeds were incubated with 1/2× MS solution for 24 h at 25°C before inoculation. Surface‐sterilized Arabidopsis thaliana seeds were vernalized for 3 days at 4°C in darkness before inoculation. For inoculation, seeds were treated with a bacterial suspension at 10⁸ CFU ml⁻¹ (colony‐forming units) for 10 min and placed on filter papers for 2 min to remove any excess bacterial suspension. Then, the seeds were transferred to a flask (3 cm in diameter and 10 cm in height) filled with 15 ml of 1/2× MS semisolid agar medium with sucrose (30 g l⁻¹). Plants were grown in a growth chamber with a photoperiod of 16 h light/8 h dark, at 80 μmol photons m⁻² s⁻¹ and a constant temperature of 25°C. Fluorescence was examined after 5–10 days of incubation with a Zeiss inverted fluorescence microscope (Zeiss LSM 800 Airyscan, Carl Zeiss Microimaging Inc., NY, USA) at 510–535 nm for GFP.

To perform confocal
microscopy, ANFs were stained by ThT as an amyloid binding fluorescent
dye. A stock solution of 5 mM ThT in PBS buffer (pH 7) was prepared,
and 0.1 mL of it was added to 9.9 mL of a 2 wt % ANF dispersion to
reach a total ThT concentration of 50 μM. The dispersion was
stirred for 5 h to let the ThT bond to the ANFs and then dialyzed
in the dark to remove nonbonded ThT. The dialysate was replaced with
a fresh solution three times a day, and the absorption spectrum of
the dialysate was collected using a plate reader. The absorption spectrum
of free ThT in the water solution shows a peak at λmax = 412,
and the dialysis process was continued until no absorption signal
was detected from ThT. The ThT-stained ANFs were then utilized to
prepare aerogels for confocal microscopy. Aerogels were prepared by
ice-templating as explained in detail before but dried via freeze-drying
instead of solvent exchange to eliminate the possible effects that
acetone might have on the ThT. Confocal microscopy was performed using
a Confocal Microscope Zeiss LSM 800 Airyscan with a Plan-Apo 63×/1.40
NA Oil objective. The samples were excited using a 458 nm argon laser,
and the emission was collected between 480 and 520 nm. Images were
analyzed using the ZEN 3.4 software.