Differential and Simultaneous Visualization of Cells and Airspaces in Plant Leaves

Materials and Reagents

1. >100 ml container to hold the staining solution (see procedure A) during rotational incubation
   
2. An incubation container for staining (e.g., 1.5 ml micro tube, Petri dish)
   
3. Super frost slide glass (Matsunami Glass Ind., catalog number: S2441 )
   
4. NEO microscope coverslip, 24 x 32 mm (Matsunami Glass, catalog number: C024321 )
   
5. Parafilm M^® (Bemis Company, Inc., catalog number: PM-996 )
   
6. Pipetman p200 or 1 ml disposable transfer pipette
   
7. Silicone oil (Shin-Etsu Chemical Co., catalog number: KF96L-1.0 cs or KF96L-1.5 cs )
   Note: It is named “Dimethyl Silicone Fluid” on Shin-Etsu Chemical Co. website.
   
8. Nile Red (Invitrogen, catalog number: N-1142 )
   Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: N-1142”.
   

Equipment

1. Tube rotator or shaker
   
2. Electronic scale
   
3. Confocal microscope (Carl Zeiss Microscopy, model: LSM-780) or fluorescence microscope (Carl Zeiss Microscopy, model: Axioskop2 plus)
   
4. Sonication bath (HONDA ELECTRONICS Co., model: W-113 )
   Note: This is not absolutely necessary.
   

Procedure