Fei tecnai g2 biotwin electron microscope

Additional grids were prepared from each bacterial pellet as described above but without negative staining with PTA. The fixed grids were washed in 0.05 M Tris with 0.05 % Tween 20 (Tris-T) for 10 min followed by etching in saturated solution of sodium metaperiodate for 15 min (Stirling and Graff, 1995 (link)). Etching was done to improve the detection of pilin and lectin domains in the midst of curli fibers. To Post-etch the grids, they were rinsed in Tris-T and blocked with 5% bovine serum albumin (BSA) in Tris for 30 min. Primary antibody dilutions were prepared in Tris-T with 0.1 % BSA (TBT) as follows: 1:20 dilution for anti-pilin and anti-lectin antisera, and 1:80 dilution for anti-curli antisera. Grids were incubated in primary antibody dilution, post-blocking for 60 min. Negative control grids were incubated in TBT only. Grids were rinsed in TBT and incubated with gold-tagged secondary antibody, diluted 1:20 in TBT, for 60 min. Subsequently, the grids were washed with TBT and distilled water, dried on filter paper, and visualized on the FEI TecnaiTM G2 Biotwin electron microscope at the National Animal Disease Center/USDA, Ames, Iowa, or stored at RT in a desiccator ready to view.