Zen 2 3 sp1

Five-day-old thalli were used for observation. For BODIPY staining, thalli were incubated in 200 nM BODIPY 493/503, dissolved in water for 30 min, washed twice in water, and used for microscopic observation. For β-estradiol treatment, gemmae were grown on a 1/2 × Gamborg’s B5 medium plate for 3 days, then soaked in a liquid medium containing 20 μM β-estradiol or 0.1% (v/v) DMSO for 48 h. The samples were mounted in a 1/2 × Gamborg’s B5 liquid medium and observed using an LSM780 confocal microscope (Carl Zeiss) equipped with an oil immersion lens (63 ×, numerical aperture = 1.4) and lambda and Airyscan detectors. For spectral imaging, the samples were excited at 488 nm (Argon 488) and 561 nm (DPSS 561–10), and emissions between 482 and 659 nm were collected. For high-resolution imaging using the Airyscan detector, samples were excited at 488 and 561 nm, and the emission was separated using a BP495-550 + BP570-630 filter. The images were acquired using line scanning. Spectral unmixing and Airy processing of the obtained images were performed using ZEN2.3 SP1 software (Carl Zeiss). We calculated the circularity of the oil bodies and analyzed colocalization using ImageJ and the Coloc_2 plugin in ImageJ Fiji (Schindelin et al., 2012 (link)), respectively.

Frozen sections were fixed in methanol at −20 °C for 15 min, washed, blocked in 3% bovine serum albumin in PBS salt solution, and incubated overnight at 4 °C with the antibodies diluted in blocking solution. The antibodies used for staining cryosections are listed in Table 1 (#3, 5, 7, 13).
After washing for 3 × 5 min in 1×PBS, sections stained for Hv1 were additionally incubated for 1 h at room temperature with an appropriate secondary antibody (#16). Sections were stained with DAPI solution (Invitrogen, 1 µg/mL) to visualize nuclei and mounted with Fluoromount G (eBioScience, San Diego, CA, USA) mounting media. The specificity of the secondary antibody was verified by omitting the primary antibody from the staining procedure. Sections were examined using a LSM800 microscope and Zen 2.3 SP1 software (Zeiss, Jena, Germany).

Foliar disks (7 mm in diameter) from 3 to 4-week-old plants were prepared with a cork-borer and immersed in RL or control solutions. After 3 h, disks were placed on glass slides and observed under a scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Auto-fluorescence of chlorophyll was observed using laser emissions at 638 and 488 nm. The images were optimized by scanning in XY mode while adjusting laser transmissivity and voltage of photomultiplier tubes. Then, in XYZ mode, Z-scans were performed. Three-dimensional reconstructions of images were done using ZEN 2.3 SP1 software (Carl Zeiss). The width of the stomatal aperture was measured using ImageJ software¹. Aperture corresponded to the minor axis of an ellipse fitted on each stoma. A total of at least 140 stomata from 10 disks of 10 different plants per condition were used for average and standard error determinations.