Histochemical Staining of Silica Body in Rice Leaf Blades

Materials and Reagents

1. Rice (Oryza sativa L. cv. Nipponbare) plants were grown in a liquid medium supplemented with 1.5 mM SiO₃^2- in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μ mol^-2 s^-1). This protocol is performed with the fifth leaf blade at foliar age 5.2 (Kido et al., 2015). The foliar age “m.n” is defined as those in which the mth leaf is fully expanded and the (m+1)th leaf is under development with a length of n/10 of the fully expanded-length
   
2. Paraformaldehyde (Wako Pure Chemical Industries, catalog number: 162-16065 )
   
3. Sodium cacodylate buffer (Nacalai Tesque, catalog number: 37238-25 )
   
4. Agar powder (Nacalai Tesque, catalog number: 01028-85 )
   
5. Phosphate Buffered Saline (PBS) (Takara Bio, catalog number: T900 )
   
6. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
   
7. Molecular sieves (Sigma-Aldrich, catalog number: M6141 )
   
8. Benzene (Nacalai Tesque, catalog number: 04017-35 )
   
9. Crystal violet lactone (Tokyo Chemical Industry UK Ltd, catalog number: C0741 )
   
10. PBS tablets (Takara Bio, catalog number: T900)
    
11. Phosphate-buffered saline (PBS) (see Recipes)
    
12. 100% ethanol solution (see Recipes)
    
13. Crystal violet lactone solution (see Recipes)
    

Equipment

1. Growth chamber (Nippon Medical & Chemical Instruments, model: LH220S )
   
2. Stirrer Hotplate (Thermo Fisher Scientific, model: Fisher Scientific Isotemp )
   
3. Diaphragm vacuum pump (Leybold-Heraeus, model: Divac 2.2L )
   
4. Desiccator (SANPLATEC, model: PC-250K )
   
5. Microwave oven (Sharp Electronics, model: RE-T12 )
   
6. Paraffin dish (Greiner Bio-One GmbH, catalog number: 908177 )
   
7. Leica VT1200S vibrating blade microtome (Leica Microsystems, model: VT1200S )
   
8. Microscope slide (Matsunami Glass, catalog number: S-2123 )
   
9. Cover slip (Matsunami Glass, catalog number: C024361 )
   
10. Optical microscope (Leica Microsystems, model: DMRPX )
    
11. CCD camera (QImaging, model: Retiga EXi )
    

Procedure