Dfc425c camera

For microscopy studies, root segments were all collected at 6 DAG. For measuring the length of the RAM, ~ 5 mm root tips were cut and immersed in chloral hydrate solution at 4 °C overnight, analyzed under Axio Imager A1 microscope (Zeiss) with Nomarski optics. For measuring cross-sectional areas, root segments were fixed with 4% paraformaldehyde (w/v), 5% glutaraldehyde (v/v) in 0.05 M sodium phosphate buffer (pH 7.2) at 4 °C overnight. The fixed material was dehydrated in an ethanol series and subsequently embedded in Technovit 7100 (Heraeus Kulzer) according to the manufacturer’s protocol. Sections (5 μm) were made with a RJ2035 microtome (Leica Microsystems) stained 1.5 min in 0.05% toluidine blue O. For phloroglucinol-HCl staining, root segments were fixed as abovementioned. The fixed material was washed with 1 x PBS (sodium phosphate buffer), and directly embedded in 6% low melting agarose dissolved in 1 x PBS. Sections (50 μm) were made with a VT1000 S vibratome (Leica Microsystems), stained with 2% phloroglucinol (in 95% ethanol) for 2 min and applied with a few drops of 37% HCl. Sections were all analysed by using a DM5500B microscope equipped with a DFC425C camera (Leica Microsystems).

Cells were seeded onto coverslips coated with collagen (0.2 mg/mL, Sigma-Aldrich, Overijse, Belgium) at a density of 8 × 10⁵ cells/well and grown for 3 days. At this time, cells did not form a monolayer in order to properly distinguish them and observe peptides’ endocytosis.
Cells were incubated for 2 h at 37 °C in the dark with LRPep1-rho or LRPep2-rho (peptides coupled to rhodamine, 200 µM in culture medium for HepaRG and HUVEC, 25 µM for ACBRI376 and 1321N1); the negative control was incubated with culture medium. The use of the rhodamine as a fluorescent probe was based on our previous experience with labeled peptides [23 (link)]. Cells were rinsed two times with PBS (per liter: 8 g NaCl, 0.2 g KCl, 2.31 g Na₂HPO₄ × 12 H₂O, 0.2 g KH₂PO₄, pH 7.4). The Hoechst solution (Hoechst 33342 trihydrochloride, Fisher Scientific) prepared at 2 µg/mL in HBSS (per liter: 0.140 g CaCl₂, 0.1 g MgCl₂ × 6H₂O, 0.4 g KCl, 0.06 g KH₂PO₄, 0.35 g NaHCO₃, 8 g NaCl, 0.121 g Na₂HPO₄ × 12H₂O, pH 7.4) was incubated for 5 min to stain nuclei. Cells were rinsed two times and mounted with HBSS.
Fluorescence was observed using a Leica DM2000 microscope equipped with a light source EL 6000 and a DFC 425C camera (Leica Microsystems, Groot Bijgaarden, Belgium).

Rooted tissue culture plantlets for phenotyping assays were grown in crystal-clear polypropylene containers (1 L) with a gas-exchange filter (OS140BOX; Duchefa Biochemie). Pots were half-filled with Agroperlite (Maasmond-Westland) and watered with modified EKM medium (3 mm MES [C₆H₁₃NO₄], pH 6.6, 2.08 mm MgSO₄, 0.88 mm KH₂PO₄, 2.07 mm K₂HPO₄, 1.45 mm CaCl₂, 0.7 mm Na₂SO₄, 0.375 mm NH₄NO₃, 15 μm iron citrate, 6.6 μm MnSO₄, 1.5 μm ZnSO₄, 1.6 μm CuSO₄, 4 μm H₃BO₃, and 4.1 μm Na₂MoO₄; Becking, 1983 ). For nodulation assays, modified EKM medium (Becking, 1983 ) was inoculated with rhizobia (OD₆₀₀ = 0.025) prior to planting the shoots. For inoculation with strain S. fredii NGR234.pHC60, containers were half-filled with sterilized river sand and watered with modified EKM medium containing the bacteria at OD₆₀₀ = 0.05.
All nodules were fixed in buffer containing 4% (w/v) paraformaldehyde mixed with 3% (v/v) glutaraldehyde in 50 mm phosphate (pH 7.4). A vacuum was applied for 2 h during a total 48-h incubation. Fixed nodules were embedded in plastic (Technovit 7100; Heraeus-Kulzer) according to the manufacturer’s recommendations. Sections (5 µm) were made using an RJ2035 microtome (Leica Microsystems). Sections were stained using 0.05% (w/v) Toluidine Blue O. Images were taken with a DM5500B microscope equipped with a DFC425c camera (Leica Microsystems).

Longitudinal sections of root nodules were made from root nodules 5 weeks post-inoculation. Plant tissue was fixed and embedded in technovit 7100 as previously described [35 (link)]. Thin Sects. (5 µm) were cut using a microtome (Leica Microsystems, Germany) placed on a glass slide and stained with 0.05% Toluidine blue for imaging. Pictures were taken using a Leica DM5500B microscope coupled with a DFC425C camera (Leica Microsystem, Germany).