Materials and Reagents

1.5 ml microcentrifuge tubes
Pipette tips
Plain slides 75 x 25 mm (VWR, catalog number: 48300-026 )
Micro cover slides, square 22 x 22 mm (VWR, catalog number: 48366-067 )
Filter (0.22 μm)
DMSO (Sigma-Aldrich, catalog number: D8418 )
Propidium iodide (PI) 10 mg (Sigma-Aldrich, catalog number: P4170 )
Ultrapure water 18.2 MΩcm
PI (Propidium Iodide) stock (see Recipes)
PI-H₂O control (see Recipes)
PI-DMSO solution (see Recipes)

Equipment

Pipettes
Confocal laser scanning microscope (Olympus, model: Fluoview FV1000 )
Ultrapure water system (Elga LabWater, model: PureLab Classic )

Software

FV10ASW v.2.0c
ImageJ 2.0.0-rc-41/1.50d

Procedure

Performing the permeability test in 1.5 ml microcentrifuge tubes
1. Add 1 ml PI-DMSO solution (Recipe 3) and PI-H₂O control (Recipe 2) into different 1.5 ml microcentrifuge tubes. Put freshly obtained, similar size about 1 cm of pinnae into the tubes. Incubate at room temperature.
2. After 5 min, 30 min, 2 h, and 5 h take out the pinnae, rinse briefly in distilled water to remove residual PI, and then mount on slides to observe fluorescence under Confocal microscope.
Confocal microscopy
1. Observe the Intracellular PI fluorescence under a Fluoview FV1000 confocal laser scanning Biological Microscope (Olympus, Japan). The software FV10ASW v.2.0c and ImageJ 2.0.0-rc-41/1.50d are used to capture the images and construct Z projections. Detect chlorophyll autofluorescence using excitation and emission wavelengths of 488 nm and 687 nm, respectively, PI using excitation and emission wavelengths of 538 and 619 nm, respectively.
2. We found that after 30 min of incubation, the PI had entered the cells (Figure 1). Images were obtained from three samples.
  
  Figure 1. Permeability test for Hymenophyllum caudiculatum fronds. Hymenophyllum caudiculatum fronds were incubated with 0.1% DMSO or distilled water (control) with 1.5 mM PI. Fluorescence of the chlorophyll (left panel) or PI (right panel) is observed. The same field of view was recorded at different wavelengths.

Data analysis

Three samples were analyzed and representative results are shown in Figure 1. After 30 min of incubation with 0.1% DMSO, PI fluorescence starts being visible inside the cells and reaches the maximum after 5 h. In contrast, PI fluorescence was almost invisible after 5 h incubation of PI with ultrapure water.

Recipes

PI (Propidium Iodide) stock solution (1 mg/ml or 1.5 mM)
To prepare, add 10 ml distilled water to 10 mg PI. Store 1 ml aliquots in 1.5 ml tubes at -20 °C, protecting from light
PI-H₂O control (10 ml)
1.5 μM propidium Iodide
ultrapure water
1. In 10 ml of ultrapure water add 10 μl of 1.5 mM PI (stock solution)
2. Filter sterilize (0.22 μm) and store at room temperature
PI-DMSO solution (10 ml)
0.1% DMSO
1.5 μM propidium Iodide
1. In 10 ml of ultrapure water add 10 μl of DMSO and 10 μl of 1.5 mM PI (stock solution)
2. Filter sterilize (0.22 μm) and store at room temperature

Acknowledgments

This work was supported by grant #11130717 from FONDECYT.

Competing interests

The author declares no conflicts of interest or competing interests.

References

Garcés, M., Ulloa, M., Miranda, A. and Bravo, L. A. (2018). Physiological and ultrastructural characterisation of a desiccation-tolerant filmy fern, Hymenophyllum caudiculatum: Influence of translational regulation and ABA on recovery. Plant Biol Mar 20(2): 288-295.