Materials and Reagents

1. Liquid blocker, “PAP pen” (Science Services, catalog number: N71310 )
   
2. Microscope slides (e.g., Superfrost; Carl Roth, catalog number: 1879.1 )
   
3. Double-sided adhesive tape (e.g., Reichelt Elektronik, Tesa Sande, catalog number: 05338 )
   
4. Syringe needle (e.g., Terumo, catalog number: NN-2425R ; VWR International, catalog number: HSWA4710005525 )
   
5. Microscope cover slips (e.g., Carl Roth, catalog number: H874.2 )
   
6. Pipette tips (200 μl)
   
7. Razor blade (e.g., VWR International, Darmstadt, catalog number: 233-5224 )
   
8. Fresh Arabidopsis plant material (growth conditions as described in Babu et al., 2013)
   
9. Deionized water
   
10. 10% glycerol (e.g., Sigma-Aldrich, catalog number: 49782 )
    
11. 1% (v/v) DMSO (e.g., Carl Roth, catalog number: 7029.2 )
    
12. 0.05% (w/v) Triton-X 100 (e.g., Carl Roth, catalog number: 3051.2 )
    
13. 3.75% (w/v) para-formaldehyde (e.g., Carl Roth, catalog number: 0335.2 )
    
14. PBS buffer (pH 7.4) (e.g., Carl Roth, catalog number: 9143.2 )
    
15. Optional: 4’,6-Diamidin-2-Phenylindol (DAPI) (e.g., Carl Roth, catalog number: 6335.1 )
    
16. SR2200 staining solution (see Recipes)
    Note: SR2200 can be ordered from: Renaissance Chemicals Ltd, Unit 1 Blackwood Hall Business Park, North Duffield, Selby, UK. Contact: Howard Weaver, enquiries@renchem.co.uk
    

Equipment

1. Tweezers
   
2. Binocular (e.g., ZEISS, model: Stemi 2000 )
   
3. Adjustable pipette 20-200 μl (e.g., Gilson Pipetman-neo P200N; Carl Roth, catalog number: HY87.1 )
   
4. Vacuum pump (e.g., Vacuubrand PC2004 Vario)
   
5. Vacuum chamber (e.g., Kartell Labware 554)
   
6. Confocal laser scanning microscope with 405 nm laser and standard filter set for DAPI imaging (e.g., Leica TCS SP8) or spectral detector if double staining with DAPI is required (e.g., Zeiss LSM780NLO with 32 channel GaAsP array)
   

Procedure