Uranyl acetate alternative

To visualize the nanoparticles using transmission electron microscopy (TEM), NPs were diluted 1:100% v/v in distilled water corresponding to 0.1 mg/mL of the PLGA. An amount of 5 µL of the ATRA-PLGA was added on a 200 mesh formvar and silicone monoxide coated copper grid (Ted Pella Inc., Mountain Lakes Blvd Redding, CA, USA) and dried overnight. The grid was stained with 5 µL of Uranyl Acetate Alternative (Ted Pella Inc., Redding, CA, USA) for one minute. Excess stain was removed to yield a dry grid. Image acquisition was performed using a Hitachi H7650 transmission electron microscope (TEM) (Tokyo, Japan) operating at 100 kV with a side mounted camera.

The samples of either full-length AlIbpA_His6 or AlIbpAN12_His6 proteins (cross-linked alone or in presence of substrate proteins) were negatively stained on grids as described in [48 (link)]. Briefly, nickel grids (400 mesh, Electron Microscopy Sciences, Hatfield, PA, USA) were treated with 2% collodion solution for film formation. Right before the experiment, grids were discharged using UV during 1 min, followed by immediate addition of 5 μL protein solution for 15 s and its further removal with filter paper. This step was followed by the addition of 4 µL Uranyl Acetate Alternative (Ted Pella, Redding, CA, USA) for 15 s, after which the contrast agent was also removed with filter paper. Samples were visualized on a Libra 120 electron microscope (Zeiss, Oberkochen, Germany) at 10,000–20,000 magnification. The quantification of aggregate sizes was performed using in-house developed software [57 (link)].

Samples of non-infected cells and phiKZ-infected cells after 5, 10, 15, 20, 30, and 40 min of infection were chemically fixed using glutaraldehyde (2.5%) for 30 min at room temperature. Cells were collected by centrifugation at 5000× g at 4 °C. Then, cell pellets were washed twice with sterile PBS, postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min at room temperature, and subjected to EMbed 812 Kit (Electron Microscopy Sciences, PA, USA) embedding, according to the manufacturer’s protocol with the replacement of the 100% Propylene Oxide with the 100% Acetone. Thin sections were cut with a diamond knife (Diatome, Nidau, Switzerland) on a ultramicrotome Ultratome III 8800 (LKB, Bromma, Sweden), transferred to nickel grids (400 mesh, Merck, Darmstadt, Germany), covered with collodion, 2% in Amyl Acetate (Electron Microscopy Sciences, PA, USA). Sections were contrasted with gadolinium triacetate (Uranyl Acetate Alternative, TedPella, Redding, CA, USA). Electron microscopy studies were performed using the Libra120 120 kV transmission electron microscope (CarlZeiss, Oberkochen, Germany) at magnification 8000–16,000×.