Materials and Reagents

1. Sterile 9 cm square Petri dishes for plant growth media
   
2. Cover slips No 1.5 (VWR International, catalog number: 631-0125 )
   
3. Microscope slides with frosted end (VWR International, Superfrost^®, catalog number: 631-0114 )
   
4. Secure Seal Adhesive Sheets (0.12 mm thick) (Grace Bio-Labs, Inc., catalog number: 620001 )
   
5. Arabidopsis lines expressing a protein of interest tagged with PA-GFP (Patterson and Lippincott-Schwartz, 2002)
   Note: In our case, for measuring histone exchange dynamics in the root stem cells we placed the H2B-PAGFP construct under the control of a root cell-specific promoter (pSCR) (Rosa et al., 2014), which derives expression simultaneously in quiescent centre cells, endodermis/cortex initials, and root endodermis. This setup allowed us to compare the dynamics of a histone protein in pluripotent stem cells as they progress into more differentiated states. In this protocol we will not mention particular cells or tissues as the procedure can be applied to any cell type in the root.
   
6. Murashige & Skoog Basal Medium with Vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories^®, catalog number: M519 )
   
7. 10% bleach (contains 5-10% sodium hypochlorite) in dH₂O (Vortex, Procter & Gamble)
   
8. NH₄NO₃ (pH 5.8) (ForMedium^TM)
   
9. Sucrose (Sigma-Aldrich)
   
10. Phytagel (Sigma-Aldrich, catalog number: P8169 )
    
11. Murashige and Skoog medium (see Recipes)
    

Equipment

1. Plant growth chamber
   
2. Two-photon photoactivation experiments were performed on an Ultima two-photon laser scanning microscope (Buker Coopration, Prairie Technologies, model: Ultima 2-Photon Microscope )
   Note: Live images were acquired using Olympus 60x 0.9 NA water immersion objectives at 512 x 512 resolution (0.203 μm/pixel) and 1 μm focal steps.
   
3. Laminar flow cabinet
   

Software

1. ImageJ software
   
2. MS Excel
   
3. Graphpad prism software
   

Procedure