Leaf material was cryo-fixed at a rate of 20,000 Kelvins /sec using a high pressure freezer unit (Leica Microsystems EM HPM100). The second step of freeze substitution of cryo-fixed samples was performed in an EM AFS unit (Leica Microsystems) at −85°C for 48 hours in 0.5% uranyl acetate in dry acetone. The samples were then infiltrated at low temperature in Lowicryl HM20 resin (Polysciences) and polymerized with a UV lamp.
For the immunogold labelling, gold grids carrying ultrathin sections (60 – 90 nm) of leaf tissue embedded in HM20 were treated using different rabbit primary antibodies against: cyanobacterial Rubisco from Se PCC6301; tobacco Rubisco; and CcmM35 (produced by Cambridge Research Biochemicals). A secondary goat polyclonal antibody to rabbit IgG conjugated with 10 nm gold particles (Abcam, UK) was used for the labeling.
Images were obtained using a transmission electron microscope (Jeol 2011 F) operating at 200kV, equipped with a Gatan Ultrascan CCD camera and a Gatan Dual Vision CCD camera.
For the immunogold labelling, gold grids carrying ultrathin sections (60 – 90 nm) of leaf tissue embedded in HM20 were treated using different rabbit primary antibodies against: cyanobacterial Rubisco from Se PCC6301; tobacco Rubisco; and CcmM35 (produced by Cambridge Research Biochemicals). A secondary goat polyclonal antibody to rabbit IgG conjugated with 10 nm gold particles (Abcam, UK) was used for the labeling.
Images were obtained using a transmission electron microscope (Jeol 2011 F) operating at 200kV, equipped with a Gatan Ultrascan CCD camera and a Gatan Dual Vision CCD camera.