1400 transmission electronmicroscope

The two-dimensional lipid monolayers were prepared using E. coli polar lipids (Avanti) as previously described30 (link). Briefly, 0.2 μg of lipids (per well) were floated on storage buffer (lacking EDTA) in a custom-made Teflon block and placed for 1 h in a humid chamber to let the chloroform evaporate. Electron microscopy grids were placed on the monolayers, followed by sequential additions and incubations of 0.1–1 μM FtsA (40 min), 4 mM ATP (20 min), 5 μM FtsZ (5 min) and 4 mM GTP (20 min) in a total volume of 90 μl. The grids were removed and negatively stained with 1% uranyl acetate as previously described28 (link). Grids were inspected and photographed with JEOL 1400 Transmission Electron Microscope coupled with a Gatan Orius CCD camera. Protofilament spacing was measured with boxes or lines using the Plot Profile tool in ImageJ.

The organoids were analyzed for the presence of organized collagen fibril structures by TEM as described before45 . Briefly the tissues were fixed in 4% paraformaldehyde, 2.5% gluteraldehyde, and 0.1 M sodium cacodylate pH7.4 in 8.0 mM CaCl2. After treatment with 1% osmium tetroxide, the tissues were dehydrated in serial dilutions of ethanol and then propylene oxide. The tissues were embedded in a mixture of Embed 812, nadic methylanhydride, dodecenyl succinic anhydride and DMP-30 (Electron Microscopy Sciences, Hatfield, PA). Sections (80 nm thick) cut with a Leica ultramicrotome, were stained with 2% aqueous uranyl acetate and 1% phosphotungstic acid, pH 3.2. The sections were viewed at 80 kV with a JEOL 1400 transmission electron microscope and a Gatan Orius widefield side mount CC Digital camera (Gatan Inc.).