Protein A—immunogold electron microscopy

To evaluate exactly the accumulation level of GDC-P in mitochondria of M and BS cells, a quantitative immunogold labeling study was made under an electron microscope. Small leaf segments (one leaf per plant) were fixed and embedded in Lowicryl K4 M resin (Chemische Werke Lowi GmbH, Waldkraiburg, Germany) as described by Ueno (1992). Ultrathin sections on Formvar-coated grids were immunolabeled with the antiserum against GDC-P and InnovaCoat Gold − 20 nm protein A nanoparticle conjugate (Innova Biosciences, Cambridge, England, UK), stained with lead citrate, and viewed under a transmission electron microscope as described by Ueno (1992). As a negative control, the antiserum was replaced by non-immune serum.

The density of GDC-P labeling was determined for mitochondria and other intracellular locations by counting the gold particles on electron micrographs at 20,000× magnification and calculating the number of particles per unit area (μm⁻²) with Image J software. We examined 13–18 mitochondria of palisade M cells and 20 mitochondria of BS cells in several sections per leaf. The density of labeling was calculated as the mean of 3 plants.