Materials and Reagents

1. Fluorescent dyes:
   1. 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein acetoxymethyl (BCECF, AM) (Invitrogen, catalog number: B-1170 ; TEFLabs, catalog number: 0062 )
      
   2. Potassium-binding benzofuran isophthalate acetoxymethyl ester (PBFI, AM) (Life Technologies, Invitrogen^TM, catalog number: P-1267MP ; TefLabs, catalog number: 0021 )
      
      
2. Other materials:
   1. Pluronic F127 (Life Technologies, Invitrogen^TM, catalog number: P-3000MP ; Teflabs, catalog number: 2510 )
      
   2. Gramicidin (Sigma-Aldrich, catalog number: G5002 )
      
   3. 1/2 MS plates containing 0.8% sucrose, 0.8% agar pH 5.7 for seedling germination and growth
      
   4. 1/10 strength MS medium (without sucrose) for dye loading
      
   5. Sterile Arabidopsis seeds of choice
      Note: This method is optimized for Arabidopsis.
      
   6. Small culture dishes (35 mm x 10 mm), small glass beaker (vol. 10 ml or smaller) or other small vessel for seedling incubation. An Eppendorf tube will work but caution should be taken when adding or removing seedlings from these tubes causes damage. We prefer a vessel with a larger opening for easier access to seedlings.
      Note: For PBFI loading, a sterile culture vessel is recommended because of the long incubation time.
      
   7. Micropore tape (3 M, catalog number: 1530-1 )
      
   8. Dye loading medium (see Recipes)
      
   9. In situ calibration buffer for BCECF (see Recipes)
      
   10. In situ calibration buffer for PBFI (see Recipes)
       

Equipment

1. Confocal or epifluorescence microscope with appropriate filters (see below for spectral characteristics of the dyes)

Software

1. Open source software ImageJ (http://rsbweb.nih.gov/ij/index.html)

Procedure