Materials and Reagents

1. 0.2 μm sterile syringe filter (Corning, catalog number: 431219 )
   
2. Microscope slide and coverslip
   
3. Needle (30 gauge)
   
4. 3 mm Whatman filter paper (GE Healthcare, Dharmacon, catalog number: 1006090 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 1006090”.
   
5. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
   
6. Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
   
7. 25% glutaraldehyde (Junsei, catalog number: 273721250 )
   
8. Sodium carcodylate (Ted Pella, catalog number: 18851 )
   
9. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5379 )
   
10. Ethanol (Merck Millipore Corporation, Calbiochem^®, catalog number: 1.00983.1011 )
    
11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 30620 )
    
12. Osmium tetroxide (OsO₄) (Ted Pella, catalog number: 96049 )
    
13. Spurr resin (Ted Pella, catalog number: 183004221 )
    
14. Double end mold (Ted Pella, catalog number: 10590 )
    
15. Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
    
16. EDTA (BioShop, catalog number: EDT001.500 )
    
17. Triton X-100 (Sigma-Aldrich, catalog number: T8532 )
    Note: This product has been discontinued.
    
18. 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: 32670 )
    
19. Acetic acid (Merck Millipore Corporation, Calbiochem^®, catalog number: 1.00063.1011 )
    
20. 1/2 strength Karnovsky’s fixative solution (see Recipes)
    
21. Toluidine blue O solution (see Recipes)
    
22. Fixative solution (see Recipes)
    
23. DAPI solution for developing pollen (see Recipes)
    
24. DAPI solution for mature pollen grain (see Recipes)
    

Equipment

1. Ultramicrotome (Boeckeler Instruments, model: MT990 motorized precision microtome )
   
2. Hot plate
   
3. Water bath
   
4. Incubator
   
5. Fume hood
   
6. Light and fluorescence microscope (Leica Microsystems, model: DM2500 )
   
7. Vacuum pump and vacuum jar
   
8. Glass pipette

Procedure