Propylene oxide

Electron microscopic analysis of CFSC-2G cells was essentially conducted as described before [6 (link)]. In brief, cells were fixed in 1× phosphate buffered saline (PBS) containing 3% glutaraldehyde, washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), and fixed in a solution of 1% osmium tetroxide (OsO₄) (Roth, Karlsruhe, Germany) solved in 25 mM sucrose buffer (Merck). Fixed cells were dehydrated and subsequently incubated in propylene oxide (Serva, Heidelberg, Germany) in a mixture of Epon resin (Serva) and propylene oxide (1:1) and pure Epon. Finally, ultrathin sections (70–100 nm) were prepared and packed upon Cu/Rh grids (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both Science Services, Munich, Germany). The samples were analyzed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope and depicted images were taken at magnifications of 2156× to 35,970×.

For electron microscopy, small tissue pieces were fixed in 3% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer (PBS). Then, samples were washed in PBS, post-fixed in 1% OsO4 (Roth, Karlsruhe, Germany) in 25 mM sucrose buffer (Merck, Darmstadt, Germany), and dehydrated by ascending ethanol series (30%, 50%, 70%, 90%, and 100%) for 10 min each. The last step was repeated three times. Dehydrated specimens were incubated in propylene oxide (Serva, Heidelberg, Germany) for 30 min, in a mixture of Epon resin (Serva, Heidelberg, Germany) and propylene oxide (1:1) for 1 h, and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 90 °C for 2 h. Ultrathin sections (70–100 nm) were stained with 0.5% uranyl acetate and 1% lead citrate (both EMS, Munich, Germany) to enhance the contrast. Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss, Oberkochen, Germany) transmission electron microscope.

Cells were fixed in 3% glutaraldehyde (Agar scientific, Wetzlar, Germany) for at least 4 h, washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), and embedded into 2.5% agarose (Sigma, Steinheim, Germany). agarose cubes were rinsed in 0.1 M Soerensen’s phosphate buffer. Post-fixation of cells was performed in 1% OsO₄ in 17% sucrose buffer. After fixation, cells were washed in 17% sucrose buffer and deionized water and dehydrated by an ethanol series (30, 50, 70, 90 and 100%) for 10 min each and the last step thrice. The dehydrated specimens were incubated in propylene oxide (Serva, Heidelberg, Germany) for 30 min, in a mixture of Epon resin (Sigma) and propylene oxide (1:1) for 1 h and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 37°C for 12 h and then 80°C for 48 h. Ultrathin sections (70-100nm) were cut with a diamond knife (Leica, Wetzlar, Germany) by an ultramicrotome (Reichert Ultradcut S, Leica) and picked up on Cu/Rh grids (HR23 Maxtaform, Plano, Wetzlar, Germany). Negative staining by uranyl acetate and lead citrate (all EMS, Munich, Germany) was performed to enhance TEM contrast. The specimens were viewed using a Zeiss Leo-906 electron microscope (Oberkochen, Germany), operated at an acceleration voltage of 60kV.

5 x 10⁶ primary WT PMCs or transformed PMC-306 were centrifuged and resuspended in 500 µl regular growth medium. For fixation, 3% glutaraldehyde dissolved in PBS was added for four hours. The subsequent steps for sample preparations and image acquisition were conducted by the Department of Electron Microscopy at the Institute of Pathology at the University Hospital Aachen. Briefly, the cells were embedded in 5% low-melting agarose (Sigma, #2070-OP), washed in phosphate buffer, post-fixed in 1% OsO4 (Roth, #8371.1) in 25 mM sucrose buffer and dehydrated by ascending ethanol series (30, 50, 70, 90 and 100%) for 10 min each. Last step was repeated 3 times. Dehydrated specimens were incubated in propylene oxide (Serva, #33715.01) for 30 min, in a mixture of Epon resin (Serva, #21045.01) and propylene oxide (1:1) for 1h and finally in pure Epon for 1h. Samples were embedded in pure Epon and polymerized at 37°C for 12 hours and at 80°C for 48 hours. Ultrathin sections (70-100 nm) were stained with 0.5% uranyl acetate and 1% lead citrate (both EMS, #22400, #17600) to enhance contrast. Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss) transmission electron microscope.

Shortly after dissection, liver tissue was cut in small pieces (1–2 mm) and fixed in 3% glutaraldehyde in 1× PBS. Samples were washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), post-fixed in 1% Osmium tetroxide (OsO₄) (Roth, Karlsruhe, Germany) in 17% sucrose buffer (Merck) and dehydrated by ascending ethanol series (30%, 50%, 70%, 90% and 100%) for 10 min each. The last step was repeated 3 times. Dehydrated specimens were incubated in propylene oxide (Serva) for 30 min, in a mixture of Epon resin (Serva) and propylene oxide (1:1) for 1 h and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 90 °C for 2 h. Ultrathin sections (70–100 nm) were cut with an ultramicrotome (Reichert Ultracut S, Leica, Wetzlar, Germany) using a diamond knife (Diatome Ltd., Nidau, Switzerland) and picked up on Cu/Rh g (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both from EMS GmbH, Munich, Germany). Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope. Pictures were acquired in magnifications of 6000×, 10,000×, 27,000× and 60,000×.

Kidney tissue was cut in small pieces and fixed in 3% glutaraldehyde in PBS. Samples were washed in 0.1 M Soerensen’s phosphate buffer (Merck), post-fixed in 1% OsO₄ (Roth, Karlsruhe, Germany) in 25 mM sucrose buffer (Merck) and dehydrated by ascending ethanol series (30, 50, 70, 90 and 100%) for 10 min each. The last step was repeated 3 times. Dehydrated specimens were incubated in propylene oxide (Serva) for 30 min, in a mixture of Epon resin (Serva) and propylene oxide (1:1) for 1 h, and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 90 °C for 2 h. Ultrathin sections (70–100 nm) were cut by ultramicrotome (Reichert Ultracut S, Leica) with a diamond knife (Leica) and picked up on Cu/Rh grids (HR23 Maxtaform, Plano). The contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both EMS). Samples were examined using a Zeiss Leo 906 transmission electron microscope (Carl Zeiss) operating at an acceleration voltage of 60 kV.

Gd₂O₃ nanoparticles, Gd₂O₃-dex nanoparticles, and Gd₂O₃-dex-RB nanoparticles samples were prepared in UPW (Table 2). Nanoparticles in solution were allowed to adsorb on glow discharged formvar–carbon-coated nickel grids (Maxtaform, 200 mesh, Plano, Wetzlar, Germany) for 10 min. Adhesive drops were removed with filter paper. Samples were viewed at an acceleration voltage of 100 kV using a Hitachi HT7800 (Hitachi, Düsseldorf, Germany) transmission electron microscope (TEM).
SK-MEL-28 and ASC were labeled (Table 1) and fixed in 3% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer, scratched off from the tissue plate, and embedded in 5% low-melting agarose (Sigma-Aldrich). After post-fixation in 1% OsO₄ (Carl Roth, Karlsruhe, Germany) in 25 mM sucrose buffer, samples were dehydrated by ascending ethanol series, incubated in propylene oxide (Serva, Heidelberg, Germany), and embedded in Epon (Serva). Ultrathin sections (70–100 nm) were cut and stained with 0.5% uranyl acetate and 1% lead citrate (both EMS, Hatfield, PA, USA) to enhance contrast. Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss, Oberkochen, Germany) TEM.