(All microscopy, including sectioning, silver enhancement, and immunolocalization was performed by Ms. Susan Fink and Dr. John Dunlap, The University of Tennessee, Center for Electron Microscopy).
Tissue Preparation for Immunogold Labeling
Root and stem tissues were obtained from uninoculated and BradyrhizobiumjaponicumUSDA110 inoculated plants at time points ranging from 2 days to 15 days post-inoculation. Root tissue was obtained from the root meristem, as well as the region of the root corresponding to the zone of the first emergent root hairs. Stem tissues were harvested from the shoot meristematic region of 15-day-old G. soja plants. Secondary, lateral roots were also collected at time points ranging from 2 days to 15 days post-inoculation.
Tissue samples were fixed in 1% paraformaldehyde, 1%glutaraldehyde, 0.1M sodium cacodylate buffer (pH 7.0) for 1 hour at room temperature, then washed in 1X SSC buffer (0.15M NaCl, 0.015M Na-citrate, pH 7.0) three times, 20 minutes each. Samples were then dehydrated with an initial series of ethanol (10%, 20%) for 10 minutes each at 20°C. Samples were further dehydrated in ethanol (30%, 50%, 75%, 90%, 100%) for 1 hour each at -20°C. Dehydrated samples were gradually infiltrated with LR (London Resin Co., Bershire, England):ethanol : LR White (2:1, 1:1, 1:2, 1 hour each) at -20°C, and pure LR White resin overnight at -20°C. Semi-thin sections (1mm) for silver enhancement and thin sections (100nm) for electron microscopy were prepared using a Reichert OMU3 ultramicrotome (Reichert, Vienna, Austria).
Conventional fixation of soybean nodules was performed as follows: Nodules from G. sojaplants 15 days post-inoculation were fixed in 3% glutaraldehyde in 0.5 M phosphate buffer (pH 6.8) for 1 hour at room temperature. Samples were then fixed in 2% OsO4 in 0.05M phosphate buffer (pH 6.8) for 1 hour at room temperature. Following fixation, samples were dehydrated in an acetone series (25%, 50%, 75%, 95%, 100%) for 30 minutes each at room temperature. Dehydrated samples were gradually infiltrated with Spurr resin (Electron Microscopy Sciences, Fort Washington, PA): acetone: Spurr’s (2:1) for one hour at room temperature, followed by infiltration with acetone: Spurr’s (1:2, pure Spurr’s) overnight each at room temperature. Samples were polymerized in fresh Spurr resin at 75°C for 16 hours. Following polymerization, thin sections were prepared as described above.
Silver Enhancement Microscopy
Thin sections of stem, root, and nodules were obtained from the LR White and Spurr embedded blocks using glass knives on gelatin-coated glass slides. Sections were incubated in serum blocking solution (SBS) (10% non-immune serum, goat) for 15 minutes at room temperature. A 1:3 (SBS:antibody) dilution of Protein A-purified anti-GS50 or anti-GS52 IgG antibody was added to the sample and incubated at 4°C for 1.5 hours. Slides were then washed three times with 1X TTBS Buffer (20 mM Tris, 500 mM NaCl, 0.5 % Tween-20, pH 7.0) twice for two minutes each, followed by two-2 minute washes in TBS (20 mM Tris, 500 mM NaCl, pH 7.0). Samples were then incubated with 1:10 diluted secondary antibody, goat anti-rabbit IgG gold conjugates (GAR-gold 15 nm, ER Laboratory, San Mateo, CA) for 1 hour at room temperature. Pre-immune controls were included for all tissue samples. Slides were washed with 1X TTBS, followed by TBS as described above. Air-dried, gold-labeled sections were treated with silver enhancer solution A and B (1:1) (Sigma Chemical, St. Louis, MO) for 8-10 minutes and washed with distilled water. Samples were fixed in 2.5% sodium thiosulfate for 2-3 minutes. Sections were observed under an Epifluorescent microscope with a polarizing filter (Nikon Inc., Melville, NY).
Immunogold Labeling for Electron Microscopy
Ultra-thin sections (100nm) were treated with SBS as described above for 15 minutes. Protein A-purified GS50 or GS52 antibodies were diluted 1:5 in 1X TTBS + 5% normal goat serum and added to the samples. Thin sections were incubated for 1.5 hours at room temperature, followed by 4 washes with PBS. Samples were incubated with 1:50 diluted secondary antibody[goat anti-rabbit IgG gold conjugates (GAR-gold 15 nm)] for 1 hour at room temperature. Samples were washed 4 times in PBS, 10 minutes each. Samples were stained for 45minutes with 2% aqueous uranyl acetate and for 5 minutes with lead citrate. Sections were viewed under 75kV with a Hitachi 600 transmission electron microscope.