EXAMPLE DATA SHEET

FIXATION OF NICOTIANA POLLEN GRAINS AND POLLEN TUBES
FOR TRANSMISSION ELECTRON MICROSCOPY

Obtained pollen from open flowers, immersed, air removed by percussion, anthers removed and kept at room temperature. Obtained pollen tubes by splitting style in half under appropriate fixative solution, aspirated under vacuum and kept at room temperature.

Mt stabilizing buffer: (MSB; 0.1 M Pipes, pH 6.8, 0.05 M KCl, 2 mM MgCl2, 10 mM EGTA, 1 mM EDTA, 10% DMSO, 0.2% sucrose)

Cacodylate buffer: (NaCaco · 3 H2O) 0.2M stock, pH 7.4, 1.VIII.99 SR (diluted 1:1 plus 0.2% sucrose)

VIALS:

1 & 2

3 & 4
Fixation: 3% GA (Sigma - opened 1.VIII.99) in 0.1M Caco    
Rinse buffer: (5 X 10 min/each) (0.1M Cacodylate buffer) 1.    
  2.    
  3.    
  4.    
  5.    
Fix: 1% OsO4 (buffered in 0.1M cacodylate)    
Rinse buffer: (4 X 15 min/each) 1.    
  2.    
  3.    
  4.    
Dehydration: (15 min each) in ethanol 30%    
  50%    
  70%    
  80%    
  95%    
Absolute ethanol 100% (3 X 15 min/each)
 1.    
  2.    
  3.    
Infiltration: 33% Spurr's resin (2 hr)    
 67% Spurr's resin (3 hr)
    
100% Spurr's resin (2 X 12 hr)
 1.    
  2.    
Embed in new Spurr's resin    
Polymerize for 6 to 8 hr at 70°C    

SPECIAL NOTE: Make sure that each major change or modification is noted! The reasons are numerous. Sometimes mistakes in preparation result in unexpected improvements. Also, you need good notes to improve the procedure. If you don't take notes, you will never be able to precisely repeat any of your experiments.