Materials and Reagents

1. 1 ml Syringes (Henke Sass-Wolff, catalog number: 5100.200V0 )
   
2. Needles (16 G, 40 mm) (Premier Healthcare & Hygiene, BD microlance, catalog number: 300637 )
   Note: A needle of different thickness can be used depending upon the maize variety.
   
3. Falcon tubes (15 ml)
   
4. Microcentrifuge tubes
   
5. Click iT EdU imaging kit (Life Technologies, catalog number: 10337 )
   Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: 10337”.
   
   1. 5-ethynyl- 2’-deoxyuridine (EdU)
      
   2. Wheat germ Agglutinin-Alexa Fluor 488 (WGA AF 488)
      
   3. Dimethylsulfoxide (DMSO)
      
   4. Click iT EdU reaction buffer
      
   5. Copper sulphate (CuSO₄)
      
   6. Click iT EdU buffer additive
      
   7. Hoechst 33342
6. Phosphate buffer saline (PBS, pH 7.4) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
   
7. Permeabilization reagent [e.g. 0.5% Triton X-100 in PBS (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: R37602 )]
   
8. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A8531 )
   
9. 100% Ethanol
   
10. Propidium iodide (PI) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: P1304MP )
    Note: PI is toxic and may cause skin and respiratory irritation. Hence it is good to handle PI in the fume hood with the personal safety protection.
    
11. Double distilled water
    
12. 2% BSA in PBS (pH 7.4) (See Recipes)
    
13. Propidium iodide stock solution (see Recipes)
    

Equipment

1. EdU Labeling chambers
   Note: These plastic chambers are custom made to fit in the desired sample). An image showing the details of this chamber is shown below (Figure 1B). As an alternative to the labeling chambers 50 ml Falcon Tubes can also be used to labeling the leaves by dipping the infected leaf in 10 µM EdU.
   
2. Confocal microscope (e.g. Leica Microsystems, model: TCS-SP5 )
   
3. Vacuum Pump
   
4. Rocker
   

Software

1. Leica Imaging Software (Leica)

Procedure