Maize Kernels – Fixation in FAA, Embedding, Sectioning and Feulgen Staining

Materials and Reagents

1. Maize Kernels
   At different developmental stages; kernels up to 20 days after pollination (DAP) usually fix and section well, at later stages the endosperm becomes progressively dry and hard, causing the sections to crumble and tear while sectioning on the microtome
   
2. Paraplast Plus (Pelco, catalog number: 18393 ; Sherwood Medical, catalog number: 8889-502005; or Sigma-Aldrich, catalog number: P3683 )
   
3. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283 )
   
4. Formaldehyde solution (histological grade, 37 wt.% in H₂O) (Sigma-Aldrich, catalog number: 533998 )
   
5. Ethanol (96% and absolute)
   
6. Tert-butanol (TBA) (Sigma-Aldrich, catalog number: B85927 )
   
7. Pararosaniline hydrochloride (Sigma-Aldrich, catalog number: P3750 )
   
8. Potassium metabisulfite (K₂S₂O₅) (Sigma-Aldrich, catalog number: 31268 )
   
9. Xylene (Xylenes, histological grade) (Sigma-Aldrich, catalog number: 534056 )
   
10. 5 M HCl (Merck-Millipore, catalog number: 1099110001 )
    
11. Decolorizing charcoal (BDH, GB) (Sigma-Aldrich, catalog number: 161551 )
    
12. DPX mounting medium (Sigma-Aldrich, catalog number: 0 6522 )
    
13. Formaldehyde – acetic acid – ethanol (FAA) (see Recipes)
    
14. Dehydrating solutions (see Recipes)
    
15. Schiff reagent (see Recipes)
    

Equipment

1. Glass vials (volume ~20 ml)
   
2. Glass fiber filters (Microfibre filters, Whatman GF/C)
   
3. Scalpels, razor blades
   
4. Metal forceps (use dedicated forceps for wax work, they are difficult to clean)
   
5. Disposable plastic Pasteur pipettes
   
6. Vacuum desiccator (Bel-Art, model: 420100000 )
   
7. Staining dishes (for melting Paraplast Plus) (Electron Microscopy Sciences, catalog number: 70312-21 )
   
8. Hot plate (set on low – around 68 °C), with aluminum foil tent to keep the heat inside
   
9. Alcohol burner
   
10. Plastic or metal molds
    Disposable Base Mold (Pelco, catalog number: 27147 )
    Tissue-Tek Base Mold (Sakura, catalog number: 4123 )
    
11. Tissue embedding cassette bases (Pelco, catalog number: 27168-1 ) or embedding rings (Pelco, catalog number: 27169-1 ) or Tissue-Tek Embedding Rings (Sakura, catalog number: 4151 )
    
12. Small beaker with molten Paraplast Plus (dedicated for wax work)
    
13. Tray with a thin layer of cold water (cool it by adding ice)
    
14. Ice bucket filled with ice
    
15. Flat ice packet (frozen cooling pack) or brass plate (optional)
    
16. Rotary microtome
    
17. Disposable microtome blades
    
18. Small brush
    
19. Forceps with sharp ends
    
20. Single-edged razorblade
    
21. Dark cardboard plate
    
22. Stereomicroscope
    
23. Coplin jars (Electron Microscopy Sciences, catalog number: 70316-02 )
    

Procedure