Materials and Reagents

1. 3,3-diaminobenzidine (Sigma-Aldrich, catalog number: D8001 )
   
2. 0.1 N HCl
   
3. 95% ethanol (bulked)
   
4. Glacial acetic acid (EMD Millipore, catalog number: UN2789 )
   
5. L-(+)-Lactic acid (Sigma-Aldrich, catalog number: L1750 )
   
6. Glycerol (Life Technologies, Invitrogen™, catalog number: 15514-011 )
   
7. Aluminum foil
   
8. Round plastic screens with the diameter closed to that of 15 ml tube (handmade, help the wheat leaf segments immerse in the staining solution). These plastic screens were cut from a big piece of plastic mesh that can be purchased from a local hardware store or online store (for example, Industrial Netting)
   
9. Wheat leaves at three leaf stage (~2 weeks old grown under the greenhouse conditions)
   

Equipment

1. Polyethylene 50 ml conical centrifuge tube (BD Biosciences, Falcon^®, catalog number: C2745 )
   
2. Polyethylene 15 ml conical centrifuge tube (BD Biosciences, Falcon^®, catalog number: C2750 )
   
3. Glass petri dish 100 mm with lid (Sigma-Aldrich, catalog number: CLS3160102 )
   
4. Balance
   
5. pH meter
   
6. Shaker
   
7. Vacuum
   
8. Microscope
   
9. Dessicator
   

Procedure