Here, a staining methods is exemplary described which allows assessing the morphology of the plants largest organelle - the vacuole.

To highlight the lumen of vacuoles in root cells from the model plant Arabidopsis thaliana, the dye 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF) is employed. BCECF is reasonably membrane permeable at a neutral pH, but acidifi cation reduces its permeability and ultimately leads to its distinct accumulation in the vacuolar lumen. Recommended as a highly specialized dye for vacuolar lumens, BCECF might also label other acidic compartments, such as the prevacuolar compartments.

Cultivation media and growth conditions of Arabidopsis thaliana seedlings

• Prepare Arabidopsis half strength (½) Murashige and Skoog (MS) medium using 2.15 g/L MS salts (Duchefa, NL), 1 % sucrose, and 0.5 g/L MES. Adjust the media pH with KOH to 5.7. For solid media preparation, add 0.8 % plant agar (Duchefa, NL) before autoclaving.
• Grow seedlings at 22 °C on vertically oriented plates (Greiner bio-one, D, No. 688161), sealed with Leucopore (Duchefa, NL). Use a long day regime of 16-h light and 8-h darkness.

Preparation of Dye Solution

• BCECF-AM (Life Technology, Molecular Probes, CA, USA) stock solution preparation. Solution of 10 mM in DMSO. To avoid frequent freezing and thawing, divide the stock solution into aliquots and store frozen at −20 °C. Protect the solution from light to ensure compound stability. The stock solution should be stable for at least 6 months.

Probing Arabidopsis Roots with BCECF-AM

• Prepare a multiwell plate (we suggest a 12-well plate; Greiner bio-one, No. 665180) by adding 1–1.5 mL of liquid ½ MS medium to each well.
• Supplement the medium with BCECF-AM to a final concentration of 10 μM (1:1,000 dilution). Mix gently to ensure dye dispersion in the medium.
• Carefully transfer the seedling into the dye-complemented liquid medium and incubate for 1 h in darkness (wrap the well plate in aluminum foil).
• Wash the seedlings by replacing the staining solution with ½MS medium and incubate them on an orbital shaker (80 rpm) for 5 min. The orbital shaker step is optional, but will lead to consistent staining.
• Transfer the seedlings to fresh ½ MS medium to preserve vitality until imaging. Immediate imaging is advisable.
• Visualize the stained roots by fl uorescent or confocal microscopy (excitation 488 nm; emission 520 nm).

https://pubmed.ncbi.nlm.nih.gov/25408446/.