2.5. Transmission Electron Microscopy (TEM)

The protocol for cryofixation, freeze-substitution, sectioning, immunolabeling, and viewing on the TEM followed Wilson and Bacic (2012), with minor alterations. A Leica EMPACT2 high pressure freezer (Leica Microsystems) was used to cryofix protoplasts at a concentration of 1 × 10⁵ protoplasts/mL at a 0, 1, 2, 4, and 24 h regeneration time and 7-day old cells. A Leica AFS2 freeze substitution unit (Leica Microsystems) was used for freeze substitution with 0.1% uranyl acetate in acetone for 48 h at −90 °C, before warming up to −50 °C. The samples were washed in acetone at −50 °C, followed by low temperature embedding in Lowicryl HM20 (Electron Microscopy Sciences). Thin sections were cut on a Leica Ultracut R microtome (Leica Microsystems), followed by immunolabeling, as described above. Images were taken using either a Philips CM120 BioTWIN or a Tecnai G2 Spirit transmission electron microscope (Thermofisher Scientific, formerly FEI).