Materials and Reagents

1. Lily, tobacco or Arabidopsis pollen tubes
   Note: Generally, mature lily and tobacco pollen grains are harvested 1-2 days after they are completely released from the anthers of flowers (Wang et al., 2010). For Arabidopsis, day-0 flowers are used for pollen collection (Boavida and McCormick, 2007).
   
2. 1% glutaraldehyde
   
3. 0.1% pectinase (Worthington Biochemical, catalog number: LS004298 )
   
4. 1% cellulose (EMD Millipore, catalog number: 219466 )
   
5. 1x phosphate buffered saline (PBS)
   
6. 0.1% NaBH₄ (INTERNATIONAL LABORATORY USA, catalog number: 1528397 )
   
7. Primary antibody
   
8. Secondary antibody
   
9. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
   
10. Sodium azide (Sigma-Aldrich, catalog number: S-2002 )
    
11. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I-8896 )
    
12. Gelatin (Sigma-Aldrich, catalog number: 48723-F )
    
13. Blocking buffer 1 (see Recipes)
    
14. Blocking buffer 2 (see Recipes)
    
15. Pollen species-specific germination media and culture conditions (see Recipes)
    

Equipment

1. Centrifuge with free angle rotor (Eppendorf, model: 5810R )
   
2. 0.22 μm syringe filter (Merck Millipore, catalog number: SLGV033NS )
   
3. Confocal microscope (Leica Microsystems, model: TCS SP8 )
   
4. Incubator
   
5. Confocal dish (cover glass-bottom dish) (Life Scientific, catalog number: SPL-100350 )
   

Procedure