To begin, obtain Nicotiana benthamiana leaves infiltrated with the agrobacterium tumefacien cells harboring desired vectors. For visualization of plasmodesmata, add an aliquot of 200 microliters of 1%aniline blue onto a microscope slide. Then, using a blade, excise the infiltrated leaf area away from the vein and transfer it to the aniline blue solution on the slide with the abaxial side up.
Ensure the leaf tissue sample is fully submerged before covering it with a cover glass. Place the slide with the sample in a desiccator attached to a vacuum pump. Evacuate for two minutes at less than 0.8 pascal.
Then, slowly release the pressure and incubate the slide in the dark for 30 minutes at room temperature. Now, visualize the fluorescent signal of aniline blue under a laser scanning confocal microscope.
