Materials and Reagents

1. Arabidopsis day-0 flowers according to Boavida and McCormick (2007)
   Note: Choose the last inflorescence buds opened at the top of the inflorescence stem. The stamen must not be higher than the stigma.
   
2. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F0804 )
   
3. Goat serum (Sigma-Aldrich, catalog number: G9023 )
   
4. Saponin (Sigma-Aldrich, catalog number: S4521 )
   
5. Primary antibody against plant cell wall polysaccharides/glycoproteins (http://www.plantprobes.net or http://www.ccrc.uga.edu/~carbosource) or digalactosyldiacylglycerol (DGDG) prepared in the laboratory using purified DGDG as antigen
   
6. Secondary antibodies conjugated to Alexa Fluor 488 (Life Technology, catalog number: A-11008 ) or Fluorescein isothiocyanate (FITC) (Sigma-Aldrich, catalog number: F0382 )
   
7. Arabidopsis pollen germination medium (PGM) (see Recipes)
   
8. 1x Tris-buffered saline (TBS) (see Recipes)
   
9. Fixation solution (see Recipes)
   
10. Calcium- and magnesium-free Dulbecco's phosphate-buffered saline (CMF-DPBS) (see Recipes)
    
11. Blocking buffer 1 (see Recipes)
    
12. Blocking buffer 2 (see Recipes)
    

Equipment

1. Pair of tweezers
   
2. Olympus CK2 inverted microscope
   
3. Microscope Zeiss AxiObserver Z1
   
4. Syringe Filter PVDF 33 mm 0.2 µm
   
5. Microcentrifuge (VWR^TM Galaxy 14D)
   
6. NucleoSpin^® Plasmid Binding Columns (Macherey-Nagel, catalog number: 740588.50 ), but columns of any other kits (Genejet or PureLink) may be used.
   
7. Incubators at 22 °C and 30 °C
   
8. Parafilm^®
   
9. Confocal Microscope TCS-SP2 operating system (Leica) and microscope Leica DMI6000B with a DFC450 C camera with FITC filter (absorption, 485-520 nm; emission, 520-560 nm wavelengths
   

Procedure