Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy

Materials and Reagents

1. Microscope slides (25 x 76 x 1.0 mm) and #1.5 cover glass (24 x 30 mm)
   
2. Arabidopsis seedlings expressing functional fluorescent protein fusions to CESAs, the catalytic subunits of the CSC, such as GFP:CESA3 (Desprez et al., 2007), YFP:CESA6 (Paredez et al., 2006) or tdTomato:CESA6 (Sánchez-Rodríguez et al., 2012) under the control of their native promoters
   
3. Household bleach (Clorox)
   
4. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71727 )
   
5. Murashige and Skoog (MS) basal salts (Caisson Laboratories, catalog number: MSP01 )
   
6. 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: RES0113M-B103X )
   
7. Sucrose (Fisher Scientific, catalog number: BP220 )
   
8. Agar (Sigma-Aldrich, catalog number: RES10020-A102X )
   
9. Vacuum Grease (Beckman Coulter)
   
10. 0.5x Murashige and Skoog (MS) media (see Recipes)
    

Equipment

1. Growth chamber to grow plant material (e.g., Percival Scientific, model: CU-36L5 )
   
2. Square plates 90 x 90 x 15 mm
   
3. Spinning disk confocal head (Yokogawa Electric Corporation) mounted on a motorized inverted microscope (e.g., Leica Microsystems, model: Leica DMI6000 or Zeiss, model: Zeiss Cell Observer SD ), equipped with 488 and/or 561 nm excitation lasers and a Photometrics QuantEM 512SC Camera
   

Software

1. Software operating the confocal microscope (e.g., Metamorph, Molecular Devices)
   
2. ImageJ (http://imagej.nih.gov/ij/)
   
3. MultipleKymograph and WalkingAverage plugins for ImageJ (J. Reitdorf and A. Seitz, http://www.embl.de/eamnet/html/body_kymograph.html)
   
4. Imaris (BitPlane)
   
5. Excel (Microsoft)
   

Procedure