Formvar carbon coated 200 mesh copper grids

For transmission electron microscopy (TEM) analysis of NPs and NP-Protein complexes, the TEM instrument JEOL JEM 2100 (JEOL, Italy) was used operating at 120 kV. Three microliter of each suspension were dropped on a Formvar Carbon coated 200 mesh copper grids (Agar Scientific, USA) and kept in a desiccator overnight for water evaporation. The size distribution was assessed by ImageJ/Fiji software (open-source, https://imagej.net/software/fiji) using NanoDefine ParticleSizer plugin (Thorsten Wagner, https://imagej.net/plugins/particlesizer) and the data were elaborated by Origin software (OriginLab, Northampton, MA, USA).

Formvar/carbon-coated 200 mesh copper grids (Agar Scientific) were prepared via glow discharge (10 mA, 10 s) using a Q150R ES sputter coater (Quorum Technologies). Phage suspensions (15 µl) were pipetted onto the grid surface for 30 s before removal using filter paper. Samples were stained using 15 µl of 2 % phosphotungstic acid. Excess stain was removed using filter paper and grids were air-dried. Samples were visualized using a JEOL JEM-2100Plus (JEOL) TEM and an accelerating voltage of 200 kV. Images were analysed and annotated using ImageJ (https://imagej.net/Fiji).

The cellular uptake and distribution of CNPs was qualitatively evaluated by TEM analysis. RAW 264.7 murine macrophages were incubated for 24 h with 40 µg/ml and for 48 h with 160 µg/ml of CNPs. At the end of exposure, cells were washed, detached and re-suspended in 2% Karnovsky solution. Before analysis, Karnovsky solution was removed and cells re-suspended in osmium tetroxide solution in 0.1 M cacodilate pH 7.3 for 1 h. After three washes in cacodilate, 0.05 M of 10 min each, cells were dehydrated in a series of ethanol solutions in MilliQ water (30%; 50%; 75%; 95% for 15 min each, and 100% for 30 min), incubated in absolute propylene oxide for 20 min and embedded in a solution of 1: 1 epoxy resin -propylene oxide for 90 min.
This mixture was renewed with pure epoxy resin over night at room temperature and later polymerized at 60 °C for 48 h. Ultrathin sections (50-70 nm) were obtained using Leica UCT ultramicrotome (Leica, Italy) and stained for 25 min with uranyl acetate and lead citrate for 20 min, washed and dried. Ultrathin sections were collected on Formvar Carbon coated 200 mesh copper grids (Agar Scientific, USA) and imaged by JEOL JEM-2100 h-transmission electron microscope at 120 kV (JEOL, Italy).