Materials and Reagents

Flowers at different developmental stages from an Arabidopsis inflorescence
JC-1 dye (Life Technologies, Molecular Probes^®, catalog number: T3168 )
DMSO 99.9% (Sigma-Aldrich, catalog number: D8418 )
Stock solution (10 mg/ml of JC-1 in DMSO)
Working solution (10 µg/ml of JC-1 in buffer A)
Buffer A (20 mM HEPES buffer, pH 7.2) (Sigma-Aldrich, catalog number: H3375 ) (see Recipes)

Equipment

Confocal microscope (Nikon Eclipse C1 Plus Confocal microscope, using EZ-C1 3.80 imaging software and Ti-Control)
Dissecting microscope (Nikon Corporation, model: SMZ800 )
Coverslip (18 x 18 mm)
Microscopic slide (26 x 76 mm)
1 ml insulin syringe with the 0.3 x 13 mm needle (BD)
Needle point tweezers

Software

NIH ImageJ software 1.47 for Windows (http://rsb.info.nih.gov/ij/)

Procedure

Pistils isolation (Figure 1, A-E).
Using a pair of tweezers, take flowers, at different developmental stages from an Arabidopsis inflorescence (Alvarez-Buylla et al., 2010). On a microscopic slide and under a dissecting microscope, remove sepals, petals, anthers and stigma using the needles of two 1 ml insulin syringes (0.3 x 13 mm) and make longitudinal superficial cuts on pistils at each side of the septum (Figure 1).
Pistils stain (Figure 1, F-G).
1. Submerge the pistils in 100 µl of a solution containing 10 µg/ml of JC-1 in buffer A.
2. Incubate for 30 min at room temperature without shaking. Protect from light, as the dye is photosensitive.
3. Gently wash the pistils three times with buffer A.
Sample preparation for microscopy (Figure 1, H-L).
1. On a microscopic slide under a dissecting microscope, use the needles of two 1 ml insulin syringes (0.3 x 13 mm) to dissect the pistils exposing the ovules
2. Add a drop of buffer A and cover the sample with a coverslip.
3. Immediately observe under a confocal microscope. The intensities of green (excitation/emission wavelength = 485/538 nm) and red (excitation/emission wavelength = 485/590 nm) are analyzed.
  
  Figure 1. Schematic illustration showing the steps required for embryo sac mitochondrial staining
Image analysis (Power Point 1). The ratio of red to green fluorescence of JC-1 images is calculated using NIH ImageJ software.
1. The outline of each embryo sac is delimited using the freehand tool to create a region of interest (ROI) and saved using the ROI manager tool (Analyze>tools>ROI manager>Add).
2. In the “Analyze” menu, select “set measurements” and click on area and “mean gray value”.
3. Using the image Menu, select “color” and then “split channels”.
4. Close the image in the blue channel. On the green channel, go to the analyze>tools>ROI manager and select the ROI added before. The ROI will appear on the image. Still in the ROI manager go to more>multi Measure. The result will appear in a new window.
5. Repeat this step with the image in the red channel.
6. Copy the results and paste them in an excel worksheet.
7. Calculate the red to green fluorescence ratio for each ROI.

Recipes

Buffer A
20 mM HEPES buffer (pH 7.2)
For 1 L of 1 M HEPES buffer:
Dissolve 238.3 g HEPES (free acid) in 500 ml of ddH₂O
Stir while adjusting the pH 7.2 with 0.5 N NaOH
Bring up the volume to 1 L with ddH₂O to prepare 1 L of 20 mM HEPES buffer (Buffer A)
Add 20 ml of 1 M HEPES buffer in 980 ml of ddH₂O

Acknowledgments

This protocol was adapted from Hauser et al. (2006). This work was supported by The Howard Hughes Medical Institute (HHMI), National Scientific and Technical Research Council (CONICET), National Agency for Promotion of Science and Technology (AGENCIA) and National University of Mar del Plata (UNMdP). We are grateful to the Editorial Committee of Bio-protocol for kindly inviting us to write this protocol.

References

Alvarez-Buylla, E. R., Benitez, M., Corvera-Poire, A., Chaos Cador, A., de Folter, S., Gamboa de Buen, A., Garay-Arroyo, A., Garcia-Ponce, B., Jaimes-Miranda, F., Perez-Ruiz, R. V., Pineyro-Nelson, A. and Sanchez-Corrales, Y. E. (2010). Flower development. Arabidopsis Book 8: e0127.
Hauser, B. A., Sun, K., Oppenheimer, D. G. and Sage, T. L. (2006). Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion. Planta 223(3): 492-499.
Martin, M. V., Fiol, D. F., Sundaresan, V., Zabaleta, E. J. and Pagnussat, G. C. (2013). oiwa, a female gametophytic mutant impaired in a mitochondrial manganese-superoxide dismutase, reveals crucial roles for reactive oxygen species during embryo sac development and fertilization in Arabidopsis. Plant Cell 25(5): 1573-1591.