Axioskop 20

Regenerated gametophytes were photographed using a Canon Powershot A350 mounted onto a compound microscope (Zeiss Axioskop 20) or a dissecting microscope (Leica M60). Histochemical assay for GUS activity was performed according to [28 (link)] and [29 (link)] with minor modifications. Briefly, the gametophytes were fixed in 80% ice-cold glycerol solution for 15 min. Next, they were vacuum infiltrated for 10 min with GUS staining solution [50 mM sodium phosphate buffer (pH 7.2), 0.5 mM potassium-ferrocyanide, and 1 mM 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal)], then transferred to fresh GUS staining solution and incubated at 37°C for 10 h. To enhance the contrast for GUS staining, the gametophytes were cleared with 70% EtOH to remove chlorophyll before examination with a compound microscope (Zeiss Axioskop 20). For GFP6 expression analysis, leaf epidermal tissues of both untransformed and transformed sporophytes were peeled to remove the auto fluorescent signal of the chlorophyll. GFP activity was imaged with a Zeiss Axioskop 20 (excitation filter 488 nm, dichroic mirror 510 nm, emission filter 520 nm).

Cell migration was assessed using a 48-well chemotaxis chamber (Neuro Probe, Cabin John, MD, USA) with polycarbonate membranes (pore size 8 µm; Neuro Probe; #PFB8) coated with collagen type I (20 µg/mL, Sigma-Aldrich Corp.; #CC050). Briefly, the lower wells of the chamber were filled with serum-free or 10% FBS culture medium to evaluate basal or induced hfNBM migration, respectively. TNFα pre-treated (10 ng/mL for 48 h) or untreated hfNBMs (4 × 10⁴ cells) were seeded in serum starved condition into the upper well and incubated for 6 h at 37 °C in 5% CO₂ atmosphere. After the incubation period, the migrated cells in the lower side of the membrane were fixed with absolute methanol for 15 min, washed with PBS, and stained for 30 min with 10% Giemsa solution (BioOptica, Milan, Italy; #05-12005E) in PBS. No migrated cells, in the upper part of the membrane, were removed and the filter was mounted on a glass slide for visualization. The number of migrated hfNBMs was counted in blind under an optical microscope (Zeiss Axioskop 20; Carl Zeiss S.p.A., Milan, Italy) in 10 fields for each well. Each experimental point was replicated at least six times in three independent experiments.

RBE4 cells were cultured in complete growth medium on square (22 mm × 22 mm) coverslips at 8 × 10⁴ cell density. The steps related to starvation and treatment are the same as those described above.
After US stimulation, the specimens were fixed with 0.5% paraformaldehyde for 10 min at RT and subsequently washed twice in PBS.
The quantitative analysis of intercellular spaces was performed on RBE4 cells using a routinely used Papanicolaou staining, without the use of the Orange G6 step, in order to obtain the greatest contrast between the stained cells and the background.
Finally, the specimens were mounted on microscope glass slides with Canada balsam. All reagents used were purchased from Sigma Diagnostics (St. Louis, MO, USA). Each sample was examined by an optical microscope (Zeiss Axioskop 20; Carl Zeiss S.p.A., Milano, Italy) at different magnifications, and images were acquired with a digital photo camera (Truechrome HD, TiEsseLab S.r.l., Milano, Italy). The intercellular spaces were measured by ImageJ software (ImageJ, National Institute of Health, Bethesda MD, USA, http://imagej.nih.gov/ij, 1.53f), converting the image in black and white, and using the threshold mode, as reported in Figure 1.
Five microscopic fields were randomly selected for each experiment at different time points; each time point was performed in triplicate.