Materials and Reagents

1. Leaves of 3-4 weeks old wild type or Nup gene silenced N. benthamiana
   Notes:
   a.This method can be applied for other dicot plants with relatively soft leaves.
   b.For Virus-induced gene silencing (VIGS) of N. benthamiana. See Zhang and Liu (2014).
   
2. 10 µM 48-mer oligo d(T) 5’-labeled with Alexa Fluor-488, HPLC-purified [purchased from custom oligo services (e.g. Eurofins Genomics)]
   Note: Dissolved in TE (Tris-EDTA) buffer and store at -80 °C, shaded.
   
3. 99.8% Methanol (4 ml per sample) (Wako pure chemical, special glade, catalog number: 131-01826 )
   
4. 99.5% Ethanol (5 ml per sampl) (Wako pure chemical, special glade, catalog number: 057-00456 )
   
5. 99.5% Ethanol/Xylene (1:1 v/v, 1 ml per sample) (Wako pure chemical, special glade, catalog number: 244-00086 )
   
6. 99.8%Methanol/Fixation solutionB (1:1 v/v, 1 ml per sample)
   
7. PerfectHyb Plus Hybridization Buffer (2 ml per sample) (Sigma-Aldrich, catalog number: H7033 )
   
8. Fixation cocktail (see Recipes)
   
9. Fixation solution A (see Recipes)
   
10. Fixation solution B (see Recipes)

Equipment

1. 3 ml vial containers
   
2. Rotary shaker (e.g. BioCraft, model: BC-730 )
   
3. Hybridization oven or incubator (50 °C) with rotary shaker (e.g. TAITEC, model: BR-23FP MR )
   
4. Confocal laser-scanning microscope (preferred) or ordinary fluorescent microscope with the filter set for GFP or Alexa Fluor-488 (e.g. Olympus, model: FV1000-D )

Procedure