Azide-modified amino acids and AF488-Alkyne were purchased from Jena Bioscience (Jena, Germany). Atto-514-Alkyne was purchased from ATTO-TEC (Siegen, Germany) and the Click-iT cell reaction buffer kit from Invitrogen (Darmstadt, Germany).
To visualize PGN in Physcomitrella patens, protonema cells were incubated in BCD medium with ADA (0.25 mM) for 22 h. The cells were washed once with BCD medium and incubated in BCD medium mixed with a Click-iT cell reaction cocktail (1:1) containing Atto-514 (1–5 µM) for 2 h. Cells were washed once with the medium prior to imaging with a confocal microscope.
For click chemistry reactions of A. thaliana and N. benthamiana, adult plants leaves were infiltrated with 1/2 MS medium containing 0.25 mM azide-modified D-amino acids or 0.125 mM azide modified L-amino acids and incubated for 24–48 h. Infiltrated leaves were either cut off (A. thaliana) or partially cut out (N. benthamiana) and incubated in ½ MS medium mixed with Click-iT cell reaction cocktail (1:1) containing Atto-514 or AF488 (1–5 µM) for 1.5–2 h. The leaves were washed once with the medium prior to imaging. In the experiments with A. thaliana seedlings, all incubation and washing steps were performed as a whole.
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