Gatan ultrascan camera

Transmission electron microscopy was performed to confirm the isolation of the NuMat by nuclease digestion and salt extraction. Sections of day 5 PSGs were made by fixing samples in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 h at 4^o C, and washing with PBS (phosphate buffer saline) for 4 times (each wash for 1 h). Post fixation was carried out in aqueous osmium tetroxide for 3 h. Washes were performed 6 times (each for 1 min) with deionized distilled water. The samples were dehydrated in a series of graded alcohols, infiltrated, and embedded in Araldite resin. Incubation was done at 80^o C for 72 h for complete polymerization. Ultra-thin (60 nm) sections were made with a glass knife on ultra-microtome (Leica Ultra cut UCT-GA-D/E-1/00), mounted on copper grids, and stained with saturated aqueous uranyl acetate (UA) and counterstained with Reynolds lead citrate (LC) [36 ]. JEOL JEM-2100 Electron Microscope with GATAN ULTRASCAN camera, charged coupled detector and digital micrograph software was used for TEM Imaging. TEM imaging was carried out at Ruska Labs, Acharya N. G. Ranga Agricultural University, Hyderabad, Telangana.

The supporting analysis with TEM provided high-resolution images of the peptide/metal structures analyzed by MS (Appendix AFigure A2) and X-ray spectromicroscopy (Appendix BFigure A6). TEM was performed on a parallel series of samples taken at fixed time-points from the same aliquots as those analyzed by MS. A JEOL 2011 LaB6 was used, operating at 200 kV with a GATAN ultrascan camera. Where required, uranyl acetate was used for contrast. TEM was also carried out for the samples that had already been analyzed by STXM. For this, a JEOL 1230 microscope operating at 100 kV was used, and no dyes or contrast agents were introduced. STXM was carried out prior to the TEM analysis to prevent electron beam induced changes to sample chemistry.