The samples were rinsed 2×10 minutes in 0.1 M sodium cacodylate buffer prior to post-fixation (2% OsO4 in 0.1 M sodium cacodylate buffer) for 1 hour, and rinsed 5×10 minutes in distilled water before bulk staining with 1.5% uranyl acetate [(CH3COO 2UO2·2H2O] in distilled water for 30 minutes in the dark.
Nine, 10 and 11 h old trochophores were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30 min then washed in the same buffer prior to postfixing in 1% osmium tetroxide (OsO4) in 0.1 M sodium cacodylate buffer.
50 μg/well underwent electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) mini gel (Pierce, Rockford, IL) after dilution in 1× Laemli buffer prior to denaturation at 95°C for 10 minutes.
RBCs were lysed with ammonium chloride buffer prior to staining.
Parallel sets of floral buds/complete flowers at equivalent stages were treated with a) 1 M sodium pyruvate (Sigma-Aldrich) in MES-KCl buffer for 30 min, or b) 500 μM SNP (Sigma-Aldrich) in MES-KCl buffer prior to the treatment whit DCFH2-DA as above.
Samples were washed in Wash buffer (140 mM NaCl, 5 mM KCl, 12 mM sodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6.0) by centrifugation at 860 g for 5 min. The platelet pellet was resuspended in resuspension buffer prior to flow cytometric analysis on a FACSCalibur (Becton Dickinson).
Protease inhibitors were added to all buffers excluding the urea buffer prior to use.
Deglycosylated ovalbumin was buffer exchanged into aqueous AmAc buffer prior to CIP dephosphorylation.
2× Laemmli sample buffer was added and the samples were heated at 100 °C for 10 min prior to sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Protein samples were stored in buffer B, but were exchanged into sodium-free buffers prior to structural and functional studies.
