Effect of fixation time, TDE concentrations and incubation time.

Cotyledons of 5 day-old seedlings ubiquitously expressing nuclear-localized tdtomato (yellow) were either not fixated (a, e, i, m, q), fixated for one hour (b, f, j, n, r) or fixated overnight (c, d, g, h, k, l, o, p, s, t) in 4% PFA solution. Afterwards samples were incubated for one hour (a-c, e-g, i-k, m-o, q-s) or overnight (d, h, l, p, t) in the respective embedding medium: 10% glycerol (a-d); 20% TDE (e-h); 50% TDE (i-l); 70% TDE (m-p); 95% TDE (q-t). Overview of leaf surface shown as inlay. Autofluorescence excited by 405 nm laser line shown in green, autofluorescence of chlorophyll excited by 552 nm laser line shown in red. Scale bars = 10μm.