Clearing and Staining Plant Tissues

ClearSee solutions were prepared by mixing xylitol powder [#04; final 10% (w/v)], sodium deoxycholate [#07; final 15% (w/v)] and urea [#19; final 25% (w/v)] in water. Seedlings, leaves and pistils of A. thaliana and gametophores of P. patens were fixed with 4% (w/v) PFA for 30-120 min (seedlings, 30 min; leaves, 120 min; pistil or gametophores, 60 min) in PBS under vacuum (∼690 mmHg) at room temperature. Fixed tissues were washed twice for 1 min each in PBS and cleared with ClearSee at room temperature for 4 days to 4 weeks or more, depending on tissue type. The minimum incubation times for clearing were 4 days for leaves, roots and moss, 7 days for seedlings, 2 weeks for pistils, and 4 weeks for mature stems. In the case of pistils, incubation for 4 weeks improved clarity. ClearSee-treated samples could be stored at room temperature for at least 5 months. For post-staining, cleared tissues were stained with Calcofluor White (final 100 µg/ml) in ClearSee solution for 1 h, and Hoechst 33342 (final 10 µg/ml) in ClearSee solution overnight. After staining, tissues were washed in ClearSee for 1 h.