Zen 2011 sp3

For imaging the Confocal Laser Scanning Microscopes LSM700 and LSM710 from Zeiss were used and gratefully provided by the microscopy core facility of SCI-TReCS (Spinal Cord Injury and Tissue Regeneration Center Salzburg). Images were taken with the ZEN 2011 SP3 or SP7 (black edition) software (Zeiss). Images were taken as confocal z-stacks using 20x, 40x, 63x oil magnification with 0.5 or 0.6 zoom and combined to merged maximum intensity projections. For qualitative analysis 2–3 animals per group were immunohistologically stained and analyzed. For quantitative analysis 5 animals per group were stained and analyzed. All images were edited and processed with the ZEN 2012 (blue edition) software (version 1.1.2.0) and Microsoft PowerPoint. 3D Render was performed using Imaris Software (version 9.1.2, Bitplane).

LjCSyGT stop-less cDNA was cloned into pDONR™221 using the primer pairs, 23 and 24’. LjCSyGT cDNAs were transferred from pDONR entry clones to the pK7WGR2 or pK7RWG2^{53 (link)} vector through an LR clonase reaction, generating plasmids no. 22 and 23. Hairy roots transformed with RFP-LjCSyGT and LjCSyGT-RFP were generated using Agrobacterium rhizogenes AR1193, as described⁵² . The plasmids ER-gk and G-gk were used as ER system and Golgi apparatus markers, respectively^{54 (link)}. The localisation of RFP-LjCSyGT or LjCSyGT-RFP was examined with a confocal microscope (LSM710, Carl Zeiss) using an EC Plan-Neofluar objective lens. RFP fluorescence was excited at 543 nm and emission was detected at 548–680 nm. GFP fluorescence was excited at 488 nm and emission was detected at 493–538 nm. Microscopic images were taken with LSM710 (Carl Zeiss) and ZEN2011 SP3 (Carl Zeiss) and analysed with ZEN2.3 SP1 (Carl Zeiss).
Hairy roots of mutant lines transformed with either RFP-LjCSyGT or LjCSyGT-RFP were harvested and extracted as described previously for LC–MS analysis. Two individual transformants with the same construct were combined to obtain sufficient sample.