Materials and Reagents

1. ½X Murashige and Skoog (MS) medium (Sigma-Aldrich, catalog number: M5519 )
   
2. Bacto Agar [Becton, Dickinson and Company (BD), catalog number: 2140101 ]
   
3. Arabidopsis thaliana seedlings, expressing Golgi-localized proteins fused to a fluorescent protein (e.g. 35S::ST-mRFP in Col, 35S::ERD2-GFP in Col, pGNL1:: GNL1-YFP in gnl1, pGNOM::GNOM-GFP in gnom)
   
4. Inhibitors [such as BrefeldinA (BFA) (Sigma-Aldrich, catalog number: B7651 ), monensin (Sigma-Aldrich, catalog number: M5273 ), salinomycin (Sigma-Aldrich, catalog number: S4526 )]
   
5. Dimethyl sulfoxide (DMSO) (Wako Chemicals USA, catalog number: 046-2198 )
   
6. Freezing planchets type B (Ted Pella, catalog number: 39201 )
   
7. Sucrose (Wako Chemicals USA, catalog number: 196-00015 )
   
8. 1.8 ml cryovials [for example: Nunc^® cryotubes (Sigma-Aldrich, catalog number: V7884-450EA )]
   
9. Methacrylate-based resin (Lowicryl HM20 resin kit) (Electron Microscopy Sciences, catalog number: 14340 )
   
10. Sodium phosphate monobasic (NaH₂PO₄) (Sigma-Aldrich, catalog number: S8282-500G )
    
11. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S7907-100G )
    
12. Liquid nitrogen
    
13. 0.5% Formvar solution (Electron Microscopy Sciences, catalog number: 15820 )
    
14. Tween-20 (Sigma-Aldrich, catalog number: P1379-25ML )
    
15. Primary antibodies against fluorescent tag [e.g. green fluorescent proteins(GFP)]
    
16. Secondary antibodies conjugated to gold nanoparticles (5, 10, or 15 nm in diameter)
    
17. Cryo-substitution solution (see Recipes)
    
18. Lead citrate (see Recipes)
    
19. Uranyl actetate solution (see Recipes)
    
20. Phosphate-buffer saline (PBS) stock solution (10x) (see Recipes)
    
21. 0.1% Tween-20 in PBS (PBS-T-0.1%) (see Recipes)
    
22. 0.5% Tween-20 in PBS (PBS-T-0.5%) (see Recipes)
    
23. Blocking buffer (see Recipes)
    
24. HM20 resin solutions (see Recipes)
    

Equipment

1. Coverwell^® silicone imaging chambers (2.5 mm deep, 20 mm diameter) (Electron Microscopy Sciences, catalog number: 70326-16 )
2. Nickel slot grids (Electron Microscopy Sciences, catalog number: 2015-Ni )
3. 0.12-0.17 mm thick coverslips (Matsunami Glass, catalog number: C022221 )
   
4. Light Forceps (Hammacher, catalog number: HWC 118-10 )
   
5. Lab-Tek Chambered Coverglass (Thermo Fisher Scientific, catalog number: 155361 )
   
6. 0.9-1.2 mm thick Glass slides (Matsunami Glass, catalog number: S24410 )
   
7. Parafilm
   
8. 22 °C plant growing chamber
   
9. Inverted confocal Microscope (Olympus, model: FV1200 )
   Note: equipped with 473 nm or 559 nm diode laser and with water immersion 63x / 1.20 numerical aperture objectives or oil immersion 100x / 1.40 numerical aperture objectives
   
10. TIRF Microscope equipped with oil immersion CFI Apo TIRF 100x H / 1.49 numerical aperture objectives (Nikon, model: Eclipse TE2000-E with TIRF2 system)
    
11. High-pressure freezer (Leica, model: EM HPM100 or Bal-tec/RMC/ABRA Fluid AG, model: HPM 010 )
    
12. Automated freeze-substitution and low-temperature resin embedding/polymerization system (for example, Leica, model: AFS2 )
    
13. Ultramicrotome (for example, Leica, model: EM UC7 ultramicrotome )
    
14. Glass knife maker (for example, Leica, model: EM KMR3 )
    
15. Ultra 45° diamond knife or other types of wet diamond knives (Diatome AG)
    
16. Transmission electron microscope (for example, FEI, model: Tecnai 12 )
    

Procedure