Sytox green

Cell viability was assessed with fluorescein diacetate (FDA) (Sigma-Aldrich, Saint-Quentin Fallavier, France) as previously described by Jones and Senft [42 (link)]. Roots were incubated with the fluorescent probe (0.5 mg·L⁻¹) for 3 min and observed using an epifluorescence microscope (Leica DMI6000B, Wetzlar, Germany; λExcitation: 359 nm; λEmission: 461 nm).
Cytochemical staining of cellulose was carried out using Direct Red 23 (Sigma-Aldrich, Saint-Quentin Fallavier, France) as described previously [43 (link)]. Roots were incubated with the probe (0.1 mg·mL⁻¹) for 30 min in darkness. After 3 washes with distilled water, roots were observed using a confocal laser-scanning microscope (Leica TCS SP5, Wetzlar, Germany; λExcitation: 560 nm; λEmission: 570–655 nm).
Staining of exDNA was performed as described by Wen et al. [44 (link)]. Sytox Green (Invitrogen, Carlsbad, CA, USA) solution was made by 1:1000 dilution in sterile water. Mucilage was released from root tips by contact with the surface of the glass slide, and 10 μL of diluted Sytox Green were added to the sample, which was then covered with a cover slip and observed using an epifluorescence microscope (Leica DMI6000B, Wetzlar, Germany; λExcitation: 359 nm; λEmission: 461 nm).

The cell permeability assay was performed using the cell-impermeable dye Sytox Green from Invitrogen (Carlsbad, CA, USA). Briefly, cells were incubated with 30 nM Sytox Green dead cell stain for 20 min in the dark at room temperature, and then visualized with a Leica DMI3000 fluorescence microscope.

Fixation of tissues for histology was in 4% paraformaldehyde. Tissue slices(6 µm) were then processed for hematoxylin/eosin staining. To determine collagen deposition, Sirius Red and Fast Green staining was performed on paraffin sections (8 µm thick) of murine hearts. The percentage of Sirius Red positive area was used as a quantitative measure of collagen deposition. For determination of cardiac myocyte cross-sectional areas, 6 µm thick paraffin-embedded sections were prepared from murine hearts and subsequently stained with Alexa Fluor 647-conjugated wheat-germ agglutinin (WGA, Life Technologies, 1:200 dilution) for cell border determination and SYTOX Green (Life Technologies, 1:1000 dilution) for nuclei detection. Images were taken from areas of transversely cut muscle fibers by confocal microscopy. A Leica TCS SP5 II confocal microscope with a ×20 objective, laser lines 488 nm for SYTOX Green and 633 nm for WGA were used for image acquisition. The MetaMorph software (Molecular Devices) was programmed to recognize individual cells based on the WGA staining in an automated fashion. Proper thresholds were set for background and excessive fibrosis exclusion. MetaMorph’s integrated morphometry analysis tool was then used to calculate the average cell area of the cardiac myocytes.