1400 flash

For transmission electron microscopy, anthers were placed in a fixative solution of 3.7% paraformaldehyde and 2.5% glutaraldehyde, Na₂HPO₄ 0.1 M, NaH₂PO₄ 0.1 M overnight and postfixed in 1% OsO₄, Na2HPO4 0.1 M, NaH₂PO₄ 0.1 M. Anthers were dehydrated through a graded ethanol series from 30% to 100% and embedded in SPURR resin. Sections were made using an ultramicrotome Leica UC7 at 70–80 nm and poststained with acetate uranyle 5% (in ethanol), lead citrate (in NaOH). Pollen was observed using a transmission electron microscope Jeol 1400 Flash. For the genotypes pi4kα1-1, npg1-2^+/− npgr1^−/− npgr2-2^−/−, hyc1, and efop3-1^−/− efop4-2^−/−, shriveled pollen grains were selected for imaging and compared with the wild-type pollen.

After α-syn was immunoprecipitated from pars intermedia tissue (all three groups) and after kinetics of fibril formation with synthetic fragment peptides was completed, 10 µL of fragment peptide was applied to a 400-mesh Formvar-carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA) to confirm presence or absence of fibrils. Grids were incubated for 1 min and washed three times with distilled water. After air-drying, grids were incubated for 1 min in a fresh solution of 1% uranyl acetate. Grids were air-dried again and imaged via transmission electron microscopy (JEOL 1400 Flash, Japan). Pictures were acquired at an accelerating voltage of 100 kV and magnification of 40 and 80 k.

In a 1.5 ml microcentrifuge test tube, SAA1 signal peptides were incubated at 37 °C for 7 days. Peptide concentrations used were 100 μM and 500 μM using 50 mM Tris buffer (pH 8) or 50% HFIP in 25 mM Tris buffer. For peptides containing 50% HFIP, each tube was centrifuged at 14,000 rpm for 10 min prior to grid preparation. The supernatant was discarded and 100 μL of phosphate-buffered saline (pH 7.4) was added to remove the HFIP and preserve the grid integrity. For the peptide solubilized in 50 mM tris buffer (in the absence of HFIP), the centrifugation and wash steps were not applied. A small volume of the peptide solution (10 μL) was deposited on the grid. The grids utilized were 400-mesh Formvar-carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA). Grids were washed three times with distilled water after an incubation of 1 min at room temperature. Water was removed with filter paper and grids were air-dried. A fresh solution of 1% uranyl acetate (1–2 μL) was deposited on each grid for 1 min. Visualization of the grids was performed with transmission electron microscopy (JEOL 1400 Flash, Japan). Acquisition of pictures was performed with accelerating voltage of 100 kV and magnification of 40 k.